 10^14 cells ~ 100 km2 of total membranes ~250 IISER Pune main campuses

 Membrane-bound intracellular compartments

 Proteins that are secreted from the cell or located in the cellular membranes play a crucial role in
many physiological and pathological processes.
 Medically important secreted proteins include cytokines, coagulation factors, growth factors and
other signaling molecules.
 The functions of membrane proteins are diverse and include ion channel activity or transport of
other molecules across the membrane, enzymatic processes, anchoring of other proteins and
receptor signaling.
 A large fraction of the clinically approved treatment regimes today use drugs directed towards (or
consisting of) secreted proteins or cell surface-associated membrane proteins.
 A secretory protein can be defined as a protein which is actively transported out of the cell. In
humans, cells such as endocrine cells and B-lymphocytes are specialized in the secretion of proteins,
but all cells in the body secrete proteins to a varying degree.
 In addition to being a rich source of new therapeutics and drug targets, a large fraction of the blood
diagnostic tests used in the clinic are directed towards secreted proteins, emphasizing the
importance of this class of proteins for medicine and biology. The most abundant secreted proteins
include pancreatic enzymes and other digestive enzymes expressed in salivary or stomach
 One of the most important secretory organs is the liver, which produces a large number of plasma
proteins such as albumin, fibrinogen and transferrin.
 Another group of highly abundant secreted proteins belong to the defensin family and are secreted
by hematopoietic cells in the bone marrow
 Chymotrypsin-like elastase family, Carboxypeptidase in pancreas, amylase in salivary gland.
 Membrane proteins constitute one of the largest and most important classes of proteins. A
membrane protein is associated or attached to the membrane of a cell or an organelle inside the cell
and can be classified as either peripheral or integral.
 Peripheral membrane proteins are associated with the membrane by being bound to either
peripheral regions of the membrane or to integral membrane proteins, but they do not fully span
the membrane.
 Integral membrane proteins contain alpha-helical or beta-barrel structures which are hydrophobic
and therefore can span the entire lipid bilayer and are linked by extra-membranous loop regions.
 The alpha-helical integral membrane proteins form the major category of membrane proteins and
are found in all types of biological membranes and will be the main focus here.
 Their key roles as transporters and receptors explain why they represent approximately 57% of all
currently approved drug targets and hence their immense importance for the pharmacological
industry.
 • 19670 human protein-coding genes
 • 12631 (64%) genes are predicted to be intracellular
 • 5520 (28%) are predicted to have at least one membrane-bound protein product
 • 1708 genes (9%) are predicted to have at least one secreted protein product
 • 189 genes (1%) have both secreted and membrane-bound isoforms
 • Enzymes that build and modify lipids are genetically encoded but lipid composition of
biomembranes is also dependent on the food we consume
 • Very thin, quasi two-dimensional film of lipids and proteins
 • Held together by non-covalent interactions among membrane components
 • Membranes are fluid and dynamic
 Technological developments, especially in mass spectrometry and bioinformatics, have revealed
that living cells contain thousands rather than dozens of different lipids. Now, the resulting
questions are what is the relevance of each of these unique molecules for the cell and how do cells
use lipids for their vital functions?
 The answer requires an integrative approach – cellular lipidomics – which addresses first the
distribution of all lipids between the various organelle membranes and then their local organization
within each membrane. To understand lipid homeostasis and its dynamics, one has to study the
localized metabolism of lipids, their transport within and between the various membranes, and the
sensors and effectors that govern these processes.
 In terms of function, above all, we need to understand the physical behavior of complex lipid
mixtures and their effect on local protein structure, organization and function.
 Finally, in the course of evolution, many lipids and lipid metabolites have acquired key functions in
the signaling networks that wire the cell, by binding to cognate receptors and by recruiting proteins
to specific membranes.
 The hydrophobic effect
 • Stability of the H-bond network leads to exclusion of molecules that cannot form H-bonds
 The hydrophobic effect Gorter and Grendel (1925)
 • Experimentally investigated the surface area of lipids
 • Extracted lipids from red blood cells of man, dog, rabbit, sheep, guinea pig, and goat in acetone
 • Spread on a water surface and the area was measured using a Langmuir film balance
 • Measured the surface area of the red blood cells from the microscopic images.
 • Found that the surface area of the monofilms was within error exactly two times that of the cells
 • Concluded that cell membranes are made of two opposing thin molecular layers
 • Proposed that two lipid layers form a bilayer with the polar head groups pointing toward the
aqueous environment
 • The Dynamic Nature of the Plasma Membrane 133 (~105 sec)
 Cell Exterior Transverse diffusion (flip-flop)
 Flex (~10-- 9sec)
 Cytosol Lateral shift (~10-- 6sec
 The possible movements of phospholipids in a membrane.
 The types of movements in which membrane phospholipids can engage and the approximate time
scales over which they occur.
 Whereas phospholipids move from one leaflet to another at a very slow rate, they diffuse laterally
within a leaflet rapidly.
 Lipids lacking polar groups, such as cholesterol, can move across the bilayer quite rapidly.
 The Diffusion of Membrane Proteins after Cell Fusion is a technique whereby two different types of
cells, or cells from two different species, can be fused to produce one cell with a common cytoplasm
and a single, continuous plasma membrane.
 Cells are induced to fuse with one another by making the outer surface of the cells “sticky” so that
their plasma membranes adhere to one another.
 Cells can be induced to fuse by addition of certain inactivated viruses that attach to the surface
membrane, by adding the compound polyethylene glycol, or by a mild electric shock. Cell fusion has
 played an important role in cell biology and is currently used in an invaluable technique to prepare
specific antibodies.
 The first experiments to demonstrate that membrane proteins could move within the plane of the
membrane utilized cell fusion, and they were reported in 1970 by Larry Frye and Michael Edidin of
Johns Hopkins University.
 In their experiments, mouse and human cells were fused, and the locations of specific proteins
of the plasma membrane were followed once the two membranes had become continuous.
 To follow the distribution of either the mouse membrane proteins or the human membrane
proteins at various times after fusion, antibodies against one or the other type of protein were
prepared and covalently linked to fluorescent dyes.
 The antibodies against the mouse proteins were complexed with a dye that fluoresces green and
the antibodies against human proteins with one that fluoresces red.
 When the antibodies were added to fused cells, they bound to the human or mouse proteins
and could be located under a fluorescence light microscope
 At the time of fusion, the plasma membrane appeared half human and half mouse; that is, the
two protein types remained segregated in their own hemisphere (step 3, Figure 4.26 a , b ).
 As the time after fusion increased, the membrane proteins were seen to move laterally within
the membrane into the opposite hemisphere. By about 40 minutes, each species’ proteins were
uniformly distributed around the entire hybrid cell membrane (step 4, Figure 4.26 a ).
 If the same experiment was performed at lower temperature, the viscosity of the lipid bilayer
increased, and the mobility of the membrane proteins decreased.
 These early cell fusion experiments gave the impression that integral membrane proteins were
capable of virtually unrestricted movements. As we will see shortly, subsequent studies made it
apparent that membrane dynamics was a much more complex subject than first envisioned.
 Restrictions on Protein and Lipid Mobility: Several techniques allow researchers to follow the
movements of molecules in the membranes of living cells using the light microscope.
 In a technique called fluorescence recovery after photobleaching (FRAP), which is illustrated in
FIGURE 4.27 a , integral membrane components in cultured cells are first labeled by linkage to a
fluorescent dye.
 A particular membrane protein can be labeled using a specific probe, such as a fluorescent
antibody.
 Once labeled, cells are placed under the microscope and irradiated by a sharply focused laser
beam that bleaches the fluorescent molecules in its path, leaving a circular spot (typically about
1 µm diameter) on the surface of the cell that is largely devoid of fluorescence. If the labeled
FIGURE 4.26
 The use of cell fusion to reveal mobility of membrane proteins. ( a) Outline of the experiment in
which human and mouse cells were fused (steps 1–2) and the distribution of the proteins on the
surface of each cell were followed in the hybrids over time (steps 3–4). Mouse membrane
proteins are indicated by solid circles, human membrane proteins by open circles. Locations of
human and mouse proteins in the plasma membranes of the hybrid cells were monitored by
interaction with fluorescent red and fluorescent green antibodies, respectively. ( b) Micrograph
showing a fused cell in which mouse and human proteins are still in their respective
hemispheres (equivalent to the hybrid in step 3 of part a).
 In light of the proceeding, we propose an anchored membrane-protein picket model (Fig. 5) in which
transmembrane proteins anchored to the actin-based membrane skeleton effectively act as pickets
along the membrane skeleton fence.

 Such rows of pickets possibly confine the movement of phospholipids through both steric hindrance
(Saxton, 1994) and circumferential slowing.

 A major characteristic of this model is the coupling of the membrane skeleton to lipids in the outer
leaflet of the membrane by means of the membrane skeleton–anchored proteins.

 To test this model, a series of Monte Carlo simulations of the diffusion of phospholipids, including
the effects of steric hindrance and circumferential slowing caused by proteins anchored to the
membrane skeleton, were performed.

 The free area theory of lipid diffusion gives an expression for the reduction in the diffusion constant
that extends radially from the immobilized proteins

 Typical smaller 230-nm compartments present within the greater purple compartment are shown
by circles in Fig. 7 A, left (enlarged in Fig. 7 C). Instances of hops between greater compartments at
0.5–1-s intervals are apparent when the locations of gold–DOPE complexes at 81-frame intervals
were connected by straight lines (like a time-lapse trajectory at a time resolution of 2 ms) as shown
in Fig. 7 A, right.

 These results clearly indicate the double compartmentalization of the NRK cell membrane (Fig. 8).
Figure 5.

 Anchored membrane-protein picket model. The transmembrane proteins anchored to the actin
membrane skeleton meshwork effectively act as rows of pickets, and temporarily confine the
movement of phospholipids through steric hindrance and circumferential slowing (packing or
frictional) effects

 Our Monte Carlo simulations indicate that mobile particles cannot form effective diffusion barriers
or obstacles. Figure 6. A series of Monte Carlo simulations support the anchored-protein picket
model. (A) Typical trajectories of test particles (2,250 steps with 25-!s intervals) bounded by
immobile obstacles as observed in Monte Carlo simulations. Stationary barrier obstacles of set hard-
 core radius were placed along the edges of a square grid (200 nm " 200 nm) such that there were a
set number of barriers per unit cell. The effects of steric hindrance and circumferential slowing due
to anchored proteins are included. • 2006 These results indicate that in the same cell line (for both
the NRK and FRSK cases), the MSK mesh size determined by electron tomography and the diffusion
compartment size determined by the high speed single-particle tracking of a phospholipid are
similar to each other. However, between these two cell lines, both the MSK mesh and the diffusion
compartment sizes differ greatly. The similarities between the MSK mesh sizes and the diffusion
compartment sizes in cell lines that exhibit quite different distributions strongly support the MSK
fence and picket models.
 . Therefore, it is interesting to compare the distribution of the mesh size of the MSK directly
attached to the cytoplasmic surface of the plasma membrane, as determined by an EM method,
with that of the compartment size for the diffusion of membrane molecules. NRK (median size = 230
nm) and FRSK (41 nm) cell lines were selected for such a comparison because their compartment
sizes are very different (Fujiwara et al., 2002; Murase et al., 2004). This will be an interesting test for
the MSK fence and MSK-anchored transmembrane protein picket models and became possible by
obtaining the 3D reconstructed images of the MSK structure on the cytoplasmic surface of the
plasma membrane.
 • Phospholipids undergo hop diffusion within 230 nm confined regions in cell membranes
 • As a consequence, long range diffusion is a reflection of diffusion within the confined regions and
their tendency to hop across these regions
 An Overview of the Endomembrane System Under the light microscope, the cytoplasm of living cells
appears relatively devoid of structure. Yet, even before the beginning of the twentieth century,
examination of stained sections of animal tissues hinted at the existence of an extensive membrane
network within the cytoplasm. It wasn ’ t until the development of the electron microscope in the
1940s, however, that biologists began to appreciate the diverse array of membrane ‐bound
structures present in the cytoplasm of most eukaryotic cells.
 These early electron microscopists saw membrane‐bound vesicles of varying diameter containing
material of different electron density; long channels bounded by membranes that radiate through
the cytoplasm to form an interconnected network of canals; and stacks of flattened membrane‐
bound sacs. It became evident from these early electron microscopic studies and the biochemical
investigations that followed that the cytoplasm of eukaryotic cells was subdivided into a variety of
distinct compartments bounded by membrane barriers. As more types of cells were examined, it
became apparent that these membranous compartments in the cytoplasm formed different
organelles that could be identified in diverse cells from yeast to multicellular plants and animals. The
extent to which the cytoplasm of a eukaryotic cell is occupied by membranous structures is
illustrated in the electron micrograph of a maize root cell shown in FIGURE 8.1 . As we will see in the
following pages, each of these organelles contains a particular complement of proteins and is
specialized for particular types of activities.
 Thus, just as a house or restaurant is divided into specialized rooms where different activities can
take place independent of one another, the cytoplasm of a cell is divided into specialized
membranous compartments for analogous reasons.
 Keep in mind, as you examine the micrographs in this chapter, that these cytoplasmic organelles
may appear as stable structures, like the rooms of a house or restaurant, but in fact they are
dynamic compartments that are in continual flux.
 In the present chapter, we will examine the structure and functions of the endoplasmic reticulum,
Golgi complex, endosomes, lysosomes, and vacuoles. Taken together, these organelles form an
endomembrane system in which the individual components function as part of a coordinated unit.
(Mitochondria and chloroplasts are not part of this interconnected system. Current evidence
suggests that peroxisomes have a dual origin. The basic elements of the boundary membrane are
thought to arise from the endoplasmic reticulum, but many of the membrane proteins and the
soluble internal proteins are taken up from the cytosol
 The organelles of the endomembrane system are part of a dynamic, integrated network in which
materials are shuttled back and forth from one part of the cell to another. For the most part,
materials are shuttled between organelles—from the Golgi complex to the plasma membrane, for
example—in small, membrane‐bounded transport vesicles that bud from a donor membrane
compartment (FIGURE 8.2 a ).
 1 Transport vesicles move through the cytoplasm in a directed manner, often pulled by
motor proteins that operate on tracks formed by microtubules and microfilaments of the
cytoskeleton (see Figure 9.1 a ).
 When they reach their destination, the vesicles fuse with the membrane of the acceptor
compartment, which receives the vesicle’s soluble cargo as well as its membranous wrapper
(Figure 8.2 a ).
 Repeated cycles of budding and fusion shuttle a diverse array of materials along numerous
pathways that traverse the cell. Several distinct pathways through the cytoplasm have been
identified and are illustrated in the overview shown in Figure 8.2 b .
 A biosynthetic pathway can be discerned in which proteins are synthesized in the
endoplasmic reticulum, modified during passage through the Golgi complex, and
transported from the Golgi complex to various destinations, such as the plasma membrane,
a lysosome, or the large vacuole of a plant cell.
 This route is also referred to as the secretory pathway , because many of the proteins
synthesized in often carry out an elaborate restructuring of host cell membranes, most often
the ER membrane, to create new organelle‐like structures, referred to as virus factories or
viroplasm.
 Inside these virus factories, viral proteins required for replication are organized into
assembly lines for the efficient production of new viral particles. In many ways, these
factories operate like a scaled‐down version of the cellular organelles from which they are
derived, but designed for the mass production of just a handful of molecules, rather than
the thousands that the ER and Golgi complex are responsible for producing in any given cell.
 ER Secretory vesicle Golgi complex -Membrane‐bound compartments of the cytoplasm. The
cytoplasm of this root cap cell of a maize plant contains an array of membrane‐bound
organelles whose structure and function .As is evident in this micrograph, the combined
surface area of the cytoplasmic membranes is many times greater than that of the
surrounding plasma membrane.
 1 The term vesicle implies a spherical‐shaped carrier. Cargo may also be transported in
irregular or tubular‐shaped membrane‐bound carriers. For the sake of simplicity, we will
generally refer to carriers as “vesicles,” while keeping in mind they are not always spherical.
 Secretory activities of cells can be divided into two types: constitutive and regulated (Figure
8.2 b ).
 During constitutive secretion , materials are transported in secretory vesicles from their
sites of synthesis and discharged into the extracellular space in a continual manner. Most
cells engage in constitutive secretion, a process that contributes not only to the formation of
the extracellular matrix (Section 7.2 ), but to the formation of the plasma membrane itself.
 During regulated secretion , materials are stored as membrane‐bound packages and
discharged only in response to an appropriate stimulus. Regulated secretion occurs, for
example, in endocrine cells that release hormones, in pancreatic acinar cells that release
digestive enzymes, and in nerve cells that release neurotransmitters. In some of these cells,
materials to be secreted are stored in large, densely packed, membrane‐bound secretory
granules (see FIGURE 8.3 ).
 Proteins, lipids, and complex polysaccharides are transported through the cell along the
biosynthetic or secretory pathway.
 During the discussion, we will consider several distinct classes of proteins. These include
soluble proteins that are discharged from the cell, integral proteins of the various
membranes depicted in Figure 8.2 b , and soluble proteins that reside within the various
compartments enclosed by the endomembranes (e.g., lysosomal enzymes).
 Whereas materials move out of the cell by the secretory pathway, the endocytic pathway
operates in the opposite direction.
 By following the endocytic pathway , materials move from the outer surface of the cell to
compartments, such as endosomes and lysosomes, located within the cytoplasm (Figure 8.2
b ).
 The movement of vesicles and their contents along the various pathways of a cell is
analogous to the movement of trucks carrying different types of cargo along the various
highways of a city. Both types of transport require defined traffic patterns to ensure that
materials are accurately delivered to the appropriate sites.
 For example, protein trafficking within a salivary gland cell requires that the proteins of
salivary mucus, which are synthesized in the endoplasmic reticulum, are specifically targeted
to secretory granules, while lysosomal enzymes, which are also manufactured in the
endoplasmic reticulum, are specifically targeted to a lysosome.
 Vesicles form by membrane budding, during which specific membrane proteins (green
spheres) of the donor membrane are incorporated into the vesicle membrane and specific
soluble proteins (purple spheres) in the donor compartment are bound to specific receptors.
 When the transport vesicle subsequently fuses to another membrane, the proteins of the
vesicle membrane become part of the recipient membrane, and the soluble proteins
become sequestered within the lumen of the recipient compartment.
 ( b) Materials follow the biosynthetic (or secretory) pathway from the endoplasmic
reticulum, through the Golgi complex, and out to various locations including lysosomes,
endosomes, secretory vesicles, secretory granules, vacuoles, and the plasma membrane.
 Materials follow the endocytic pathway from the cell surface to the interior by way of
endosomes and lysosomes, where they are generally degraded by lysosomal enzymes
 Endoplasmic Reticulum
 • Constitutes more than half of the total membranes of the cell
 • Composed of a network of branching tubules and flattened sacs
 • ER membrane connected to the nuclear membrane and lumen connected to extracellular
space
 • ER membrane is the site of most lipid and membrane protein synthesis in the cell
 Endoplasmic Reticulum and Membrane Protein Biogenesis
 Smooth ER Rough ER Morphologically divided into two classes Endoplasmic Reticulum
Organization
 The RER is continuous with the outer membrane of the nuclear envelope, which also
bears ribosomes on its cytosolic surface (Figure 8.2 b ). In contrast, the membranes of
the SER are highly curved and tubular, forming an interconnecting system of pipelines
traversing the cytoplasm (Figure 8.9 a ). When cells are homogenized, the SER tubules
fragment into smooth‐surfaced vesicles, whereas the RER sheets fragment into rough ‐
surfaced vesicles (Figure 8.5 b , c ). Fluorescently labeled proteins and lipids are capable
of diffusing from one type of ER into the other, indicating that their membranes are
continuous. In fact, the two types of ER share many of the same proteins and engage in
certain common activities, such as the synthesis of certain lipids and cholesterol.
 At the same time, numerous proteins are found only in one or the other type of ER. For
example, the high degree of curvature of the SER tubules is induced and maintained by
the presence of large numbers of membrane‐bending proteins, called reticulons, which
are only present in small numbers at the edges of the flattened RER sheets. The RER, in
contrast, contains high levels of proteins involved in the movement of nascent proteins
into the ER lumen. Different types of cells contain markedly different ratios of the two
types of ER, depending on the activities of the cell. For example, cells that secrete large
amounts of proteins, such as the cells of the pancreas or salivary glands, have extensive
regions of RER (Figure 8.9 b–d ). We will return to the function of the RER shortly, but
first we will describe the activities of the SER. The Smooth Endoplasmic Reticulum The
SER is extensively developed in a number of cell types, including those of skeletal
muscle, kidney tubules, and steroid‐producing endocrine glands (FIGURE 8.10 ).
 SER functions include:
 ● Synthesis of steroid hormones in the endocrine cells of the gonad and adrenal cortex.
 Intracellular Compartments and Protein Sorting
 The Compartmentalization of Cells The Transport of Molecules between the Nucleus
and the Cytosol The Transport of Proteins into Mitochondria and Chloroplasts
Peroxisomes
 (A) An electron micrograph of the rough ER in a pancreatic exocrine cell that makes
and secretes large amounts of digestive enzymes every day. The cytosol is filled with
closely packed sheets of ER membrane studded with ribosomes. At the top left is a
portion of the nucleus and its nuclear envelope; note that the outer nuclear
membrane, which is continuous with the ER, is also studded with ribosomes.
 (B) A thin section electron micrograph of polyribosomes attached to the ER
membrane. The plane of section in some places cuts through the ER roughly parallel
to the membrane, giving a face-on view of the rosettelike pattern of the
polyribosomes
 The smooth ER. (A) Abundant smooth ER in a steroid-hormone-secreting cell. This
electron micrograph is of a testosterone-secreting Leydig cell in the human testis.
 (B) A three-dimensional reconstruction of a region of smooth ER and rough ER in a
liver cell. The rough ER forms oriented stacks of flattened cisternae, each having a
lumenal space 20 30 nm wide. The smooth ER membrane is connected to these
cisternae and forms a fine network of tubules 30 60 nm in diameter
 Polarized structure of a secretory cell. ( a) Drawing of a mucus‐secreting goblet cell
from the rat colon. ( b) Low‐power electron micrograph of a mucus‐secreting cell
from Brunner ’ s gland of the mouse small intestine. Both types of cells display a
distinctly polarized arrangement of organelles, reflecting their role in secreting large
quantities of mucoproteins. The basal ends of the cells contain the nucleus and
rough ER. Proteins synthesized in the rough ER move into the closely associated
Golgi complex and from there into membrane‐bound carriers in which the final
secretory product is concentrated. The apical regions of the cells are filled with
secretory granules containing the mucoproteins ready for release into a duct.
SOURCE: (a) From Marian Neutraand C. P. Leblond , Copyright 1966, Rockefeller
University Press . Originally published in The Journal of Cell Biology Volume 30:119.
(b): From Alain Rambourg and Yves Clermont , Eur. J. Cell Biol. 51 : 196 , 1990 ; ©
1990, reprinted with permission of Elsevier. Karp-c08.indd 267 11/23/2015 11:05:37
PM 8.3 • The Endoplasmic Reticulum 267 ● Detoxification in the liver of a wide
variety of organic compounds, including barbiturates and ethanol, whose chronic
use can lead to proliferation of the SER in liver cells. Detoxification is carried out by a
collection of oxygen‐transferring enzymes (oxygenases), including the cytochrome
P450 family. These enzymes are noteworthy for their lack of substrate specificity,
being able to oxidize thousands of different hydrophobic compounds and convert
them into more hydrophilic, more readily excreted derivatives. The results are not
always positive. For example, the relatively harmless compound benzo[ a ]pyrene
formed when meat is charred on a grill is converted into a potent carcinogen by the
“detoxifying” enzymes of the SER. Cytochrome P450s metabolize many prescribed
medications, and genetic variation in these enzymes among humans may explain
differences from one person to the next in the effectiveness and side effects of
many drugs. ● Sequestering calcium ions within the cytoplasm of cells. The
regulated release of Ca 2+ from the SER of skeletal and cardiac muscle cells (known
as the sarcoplasmic reticulum in muscle cells) triggers contraction. The Rough
Endoplasmic Reticulum Early investigations into the functions of the RER were
carried out on cells that secrete large quantities of proteins, such as the acinar cells
of the pancreas (Figure 8.3 ) or the mucus‐secreting cells of the lining of the
digestive tract (FIGURE 8.11 ). It is evident from the FIGURE 8.10 The smooth ER
(SER). Electron micrograph of a Leydig cell from the testis showing the extensive
smooth ER where steroid hormones are synthesized. SOURCE: Don Fawcett/Photo
Researchers, Inc. Golgi complex Nucleus Mitochondrion RER Mucigen granule (a) (b)
Secretory granules Golgi complex Lysosome Duct Rough ER FIGURE 8.11 Polarized
structure of a secretory cell. ( a) Drawing of a mucus‐secreting goblet cell from the
rat colon. ( b) Low‐power electron micrograph of a mucus‐secreting cell from
Brunner ’ s gland of the mouse small intestine. Both types of cells display a distinctly
polarized arrangement of organelles, reflecting their role in secreting large
quantities of mucoproteins. The basal ends of the cells contain the nucleus and
rough ER. Proteins synthesized in the rough ER move into the closely associated
Golgi complex and from there into membrane‐bound carriers in which the final
secretory product is concentrated. The apical regions of the cells are filled with
secretory granules containing the mucoproteins ready for release into a duct.
SOURCE: (a) From Marian Neutraand C. P. Leblond , Copyright 1966, Rockefeller
University Press . Originally published in The Journal of Cell Biology Volume 30:119.
(b): From Alain Rambourg and Yves Clermont , Eur. J. Cell Biol. 51 : 196 , 1990 ; ©
1990, reprinted with permission of Elsevier. Karp-c08.indd 267 11/23/2015 11:05:37
PM Polarity of the Endoplasmic Reticulum Recent Insights into Endoplasmic
Reticulum Organization Ref: Jonathon Nixon-Abell et al. Science (2016) ER
organization during cell division How is the ER made? Endoplasmic Reticulum:
Keeping in shape. Blackstone and Prinz (2016) eLife Reconstituting the reticular ER
network – mechanistic implications and open questions Wang and Rapoport (2019)
J. Cell Sci. Membrane tubulation Reticulons decide the shape of the ER membrane
Reticulons Membrane tubule Membrane fusion Atlastin Atlastins fuse membranes
Reconstituting the reticular ER network – mechanistic implications and open
questions Wang and Rapoport (2019) J. Cell Sci. Proof-of-concept Structure of the ER
in cells Reconstituting the reticular ER network – mechanistic implications and open
questions Wang and Rapoport (2019) J. Cell Sci. Vesicles containing reticulon (Yop1)
and atlastin (Sey1) Integral membrane protein (IMP) biogenesis Membrane Protein
Insertion at the Endoplasmic Reticulum Shao and Hegde (2011) Annu. Rev. Cell Dev.
Biol. • IMPs make up 20–30% of the eukaryotic proteome • Diverse proteins -
signaling receptors, mediate intracellular trafficking, facilitate organelle biogenesis,
and transport a variety of molecules across cellular membranes • IMPs range from
having a single transmembrane domain (TMD) that simply anchors a soluble domain
to the membrane to having tightly packed bundles containing more than 20 TMDs. •
All IMPs are translated at the ribosome and most IMPs are initially assembled at the
endoplasmic reticulum (ER) • IMP’s TMD(s) are integrated into the membrane, final
topology is determined, and tertiary and quaternary structures are achieved • If
these steps in IMP biogenesis are successful, the IMP is subsequently sorted to its
final location of function • Otherwise, one of several quality-control pathways
routes the IMP for degradation Integral membrane protein (IMP) biogenesis • IMPs
destined to be inserted into the ER face a set of challenges • The hydrophobic TMDs
of IMPs must be continuously shielded from the aqueous cytosol (shielding is
essential because the tremendously crowded cytosolic environment (~300 mg/ml
protein) would promote potentially toxic aggregation) • TMDs need to be
recognized as they emerge from the ribosome by the targeting machinery. • IMPs
must be targeted to the appropriate organelle, which requires the cytosolic
targeting factors to interface with specific membrane receptors. • TMDs need a
route of transport past the highly polar surface of the membrane into the
hydrophobic core of the lipid bilayer. • TMD insertion must be asymmetric, with the
final orientation consistent with the IMP’s final folded state. • This means that the
insertion machinery must recognize, orient, and provide a potential path into the
membrane for a wide range of sequences. Integral membrane protein (IMP)
biogenesis Co-tanslational Post-translational • Co-translational synthesis (translation
and insertion are sequential processes) • Constitutes bulk of the membrane proteins
• Synthesized and assembled at the ER • Involves the translocon apparatus • No
chaperones necessary for folding • Post-translational synthesis (translation and
insertion are temporally distinct processes) • Constitutes membrane proteins of
mitochondria and chloroplast, nuclei and peroxisome and some bacterial toxins for
e.g., alpha hemolysin, colicin, melittin and C-terminally anchored proteins found in
synaptic vesicles • Assisted (not involving the translocon apparatus)/ spontaneous
insertion into membranes • Chaperones necessary to maintain solubility in the
cytosol Integral membrane protein (IMP) biogenesis 670 Chapter 12: Intracellular
Compartments and Protein Sorting The ER Is Structurally and Functionally Diverse
While the various functions of the ER are essential to every cell, their relative
importance varies greatly between individual cell types. To meet diferent functional
demands, distinct regions of the ER become highly specialized. We observe such
functional specialization as dramatic changes in ER structure, and diferent cell types
can therefore possess characteristically diferent types of ER membrane. One of the
most remarkable ER specializations is the rough ER. Mammalian cells begin to
import most proteins into the ER before complete synthesis of the polypeptide
chain—that is, import is a co-translational process (Figure 12–32A). In contrast, the
import of proteins into mitochondria, chloroplasts, nuclei, and peroxisomes is a
post-translational process (Figure 12–32B). In co-translational transport, the
ribosome that is synthesizing the protein is attached directly to the ER membrane,
enabling one end of the protein to be translocated into the ER while the rest of the
polypeptide chain is being synthesized. Tese membrane-bound ribosomes coat the
surface of the ER, creating regions termed rough endoplasmic reticulum, or rough
ER; regions of ER that lack bound ribosomes are called smooth endoplasmic
reticulum, or smooth ER (Figure 12–33). Most cells have scanty regions of smooth
ER, and the ER is often partly smooth and partly rough. Areas of smooth ER from
which transport vesicles carrying newly synthesized proteins and lipids bud of for
transport to the Golgi apparatus are called transitional ER. In certain specialized
cells, the smooth ER is abundant and has additional functions. It is prominent, for
example, in cells that specialize in lipid metabolism, such as cells that synthesize
steroid hormones from cholesterol; the expanded smooth ER accommodates the
enzymes that make cholesterol and modify it to form the hormones (see Figure 12–
33B). Te main cell type in the liver, the hepatocyte, also has a substantial amount of
smooth ER. It is the principal site of production of lipoprotein particles, which carry
lipids via the bloodstream to other parts of the body. Te enzymes that synthesize
the lipid components of the particles are located in the membrane of the smooth
ER, which also contains enzymes that catalyze a series of reactions to detoxify both
lipid-soluble drugs and various harmful compounds produced by metabolism. Te
most extensively studied of these detoxifcation reactions are carried out by the
cytochrome P450 family of enzymes, which catalyze a series of reactions in which
water-insoluble drugs or metabolites that would otherwise accumulate to toxic
levels in cell membranes are rendered sufciently water-soluble to leave the cell and
be excreted in the urine. Because the rough ER alone cannot house enough of these
and other necessary enzymes, a substantial portion of the membrane in a
hepatocyte normally consists of smooth ER (see Table 12–2). Another crucially
important function of the ER in most eukaryotic cells is to sequester Ca2+ from the
cytosol. Te release of Ca2+ into the cytosol from the ER, and its subsequent
reuptake, occurs in many rapid responses to extracellular 5′ 5′ 3′ 3′ CO-
TRANSLATIONAL TRANSLOCATION POST-TRANSLATIONAL TRANSLOCATION MBoC6
m12.35/12.35 (A) (B) growing polypeptide chain mRNA ER-bound ribosome free
ribosome ER ER Figure 12–32 Co-translational and posttranslational protein
translocation. (A) Ribosomes bind to the ER membrane during co-translational
translocation. (B) By contrast, cytosolic ribosomes complete the synthesis of a
protein and release it prior to post-translational translocation. In both cases, the
protein is directed to the ER by an ER signal sequence (red and orange). 670 Chapter
12: Intracellular Compartments and Protein Sorting The ER Is Structurally and
Functionally Diverse While the various functions of the ER are essential to every cell,
their relative importance varies greatly between individual cell types. To meet
diferent functional demands, distinct regions of the ER become highly specialized.
We observe such functional specialization as dramatic changes in ER structure, and
diferent cell types can therefore possess characteristically diferent types of ER
membrane. One of the most remarkable ER specializations is the rough ER.
Mammalian cells begin to import most proteins into the ER before complete
synthesis of the polypeptide chain—that is, import is a co-translational process
(Figure 12–32A). In contrast, the import of proteins into mitochondria, chloroplasts,
nuclei, and peroxisomes is a post-translational process (Figure 12–32B). In co-
translational transport, the ribosome that is synthesizing the protein is attached
directly to the ER membrane, enabling one end of the protein to be translocated
into the ER while the rest of the polypeptide chain is being synthesized. Tese
membrane-bound ribosomes coat the surface of the ER, creating regions termed
rough endoplasmic reticulum, or rough ER; regions of ER that lack bound ribosomes
are called smooth endoplasmic reticulum, or smooth ER (Figure 12–33). Most cells
have scanty regions of smooth ER, and the ER is often partly smooth and partly
rough. Areas of smooth ER from which transport vesicles carrying newly synthesized
proteins and lipids bud of for transport to the Golgi apparatus are called transitional
ER. In certain specialized cells, the smooth ER is abundant and has additional
functions. It is prominent, for example, in cells that specialize in lipid metabolism,
such as cells that synthesize steroid hormones from cholesterol; the expanded
smooth ER accommodates the enzymes that make cholesterol and modify it to form
the hormones (see Figure 12–33B). Te main cell type in the liver, the hepatocyte,
also has a substantial amount of smooth ER. It is the principal site of production of
lipoprotein particles, which carry lipids via the bloodstream to other parts of the
body. Te enzymes that synthesize the lipid components of the particles are located
in the membrane of the smooth ER, which also contains enzymes that catalyze a
series of reactions to detoxify both lipid-soluble drugs and various harmful
compounds produced by metabolism. Te most extensively studied of these
detoxifcation reactions are carried out by the cytochrome P450 family of enzymes,
which catalyze a series of reactions in which water-insoluble drugs or metabolites
that would otherwise accumulate to toxic levels in cell membranes are rendered
sufciently water-soluble to leave the cell and be excreted in the urine. Because the
rough ER alone cannot house enough of these and other necessary enzymes, a
substantial portion of the membrane in a hepatocyte normally consists of smooth
ER (see Table 12–2). Another crucially important function of the ER in most
eukaryotic cells is to sequester Ca2+ from the cytosol. Te release of Ca2+ into the
cytosol from the ER, and its subsequent reuptake, occurs in many rapid responses to
extracellular 5′ 5′ 3′ 3′ CO-TRANSLATIONAL TRANSLOCATION POST-TRANSLATIONAL
TRANSLOCATION MBoC6 m12.35/12.35 (A) (B) growing polypeptide chain mRNA ER-
bound ribosome free ribosome ER ER Figure 12–32 Co-translational and
posttranslational protein translocation. (A) Ribosomes bind to the ER membrane
during co-translational translocation. (B) By contrast, cytosolic ribosomes complete
the synthesis of a protein and release it prior to post-translational translocation. In
both cases, the protein is directed to the ER by an ER signal sequence (red and
orange). 671 signals, as discussed in Chapter 15. A Ca2+ pump transports Ca2+ from
the cytosol into the ER lumen. A high concentration of Ca2+-binding proteins in the
ER facilitates Ca2+ storage. In some cell types, and perhaps in most, specifc regions
of the ER are specialized for Ca2+ storage. Muscle cells have an abundant, modifed
smooth ER called the sarcoplasmic reticulum. Te release and reuptake of Ca2+ by
the sarcoplasmic reticulum trigger myofbril contraction and relaxation, respectively,
during each round of muscle contraction (discussed in Chapter 16). To study the
functions and biochemistry of the ER, it is necessary to isolate it. Tis may seem to be
a hopeless task because the ER is intricately interleaved with other components of
the cytoplasm. Fortunately, when tissues or cells are disrupted by homogenization,
the ER breaks into fragments, which reseal to form small (~100–200 nm in diameter)
closed vesicles called microsomes. Microsomes are relatively easy to purify. To the
biochemist, microsomes represent small authentic versions of the ER, still capable of
protein translocation, protein glycosylation (discussed later), Ca2+ uptake and
release, and lipid synthesis. Microsomes derived from rough ER are studded with
ribosomes and are called rough THE ENDOPLASMIC RETICULUM nucleus inner
nuclear membrane outer nuclear membrane ER membrane ER membrane (A) 200
nm (B) (D) 200 nm 0.5 µm rough ER smooth ER ER lumen (C) MBoC6 m12.36/12.36
Figure 12–33 The rough and smooth ER. (A) An electron micrograph of the rough ER
in a pancreatic exocrine cell that makes and secretes large amounts of digestive
enzymes every day. The cytosol is filled with closely packed sheets of ER membrane
that is studded with ribosomes. At the top left is a portion of the nucleus and its
nuclear envelope; note that the outer nuclear membrane, which is continuous with
the ER, is also studded with ribosomes. (B) Abundant smooth ER in a steroid-
hormone-secreting cell. This electron micrograph is of a testosterone-secreting
Leydig cell in the human testis. (C) A three-dimensional reconstruction of a region of
smooth ER and rough ER in a liver cell. The rough ER forms oriented stacks of
flattened cisternae, each having a lumenal space 20–30 nm wide. The smooth ER
membrane is connected to these cisternae and forms a fine network of tubules 30–
60 nm in diameter. The ER lumen is colored green. (D) A tomographic reconstruction
of a portion of the ER network in a yeast cell. Membrane-bound ribosomes (tiny
dark spheres) are seen in both flat sheets and tubular regions of irregular diameter,
demonstrating that the ribosomes bind to ER membranes of different curvature in
these cells. (A, courtesy of Lelio Orci; B, courtesy of Daniel S. Friend; C, after R.V.
Krstic´ , Ultrastructure of the Mammalian Cell. New York: Springer-Verlag, 1979; D,
from M. West et al., J. Cell Biol. 193:333–346, 2011. With permission from
Rockefeller University Press.) 671 signals, as discussed in Chapter 15. A Ca2+ pump
transports Ca2+ from the cytosol into the ER lumen. A high concentration of Ca2+-
binding proteins in the ER facilitates Ca2+ storage. In some cell types, and perhaps
in most, specifc regions of the ER are specialized for Ca2+ storage. Muscle cells have
an abundant, modifed smooth ER called the sarcoplasmic reticulum. Te release and
reuptake of Ca2+ by the sarcoplasmic reticulum trigger myofbril contraction and
relaxation, respectively, during each round of muscle contraction (discussed in
Chapter 16). To study the functions and biochemistry of the ER, it is necessary to
isolate it. Tis may seem to be a hopeless task because the ER is intricately
interleaved with other components of the cytoplasm. Fortunately, when tissues or
cells are disrupted by homogenization, the ER breaks into fragments, which reseal to
form small (~100–200 nm in diameter) closed vesicles called microsomes.
Microsomes are relatively easy to purify. To the biochemist, microsomes represent
small authentic versions of the ER, still capable of protein translocation, protein
glycosylation (discussed later), Ca2+ uptake and release, and lipid synthesis.
Microsomes derived from rough ER are studded with ribosomes and are called
rough THE ENDOPLASMIC RETICULUM nucleus inner nuclear membrane outer
nuclear membrane ER membrane ER membrane (A) 200 nm (B) (D) 200 nm 0.5 µm
rough ER smooth ER ER lumen (C) MBoC6 m12.36/12.36 Figure 12–33 The rough
and smooth ER. (A) An electron micrograph of the rough ER in a pancreatic exocrine
cell that makes and secretes large amounts of digestive enzymes every day. The
cytosol is filled with closely packed sheets of ER membrane that is studded with
ribosomes. At the top left is a portion of the nucleus and its nuclear envelope; note
that the outer nuclear membrane, which is continuous with the ER, is also studded
with ribosomes. (B) Abundant smooth ER in a steroid-hormone-secreting cell. This
electron micrograph is of a testosterone-secreting Leydig cell in the human testis.
(C) A three-dimensional reconstruction of a region of smooth ER and rough ER in a
liver cell. The rough ER forms oriented stacks of flattened cisternae, each having a
lumenal space 20–30 nm wide. The smooth ER membrane is connected to these
cisternae and forms a fine network of tubules 30–60 nm in diameter. The ER lumen
is colored green. (D) A tomographic reconstruction of a portion of the ER network in
a yeast cell. Membrane-bound ribosomes (tiny dark spheres) are seen in both flat
sheets and tubular regions of irregular diameter, demonstrating that the ribosomes
bind to ER membranes of different curvature in these cells. (A, courtesy of Lelio Orci;
B, courtesy of Daniel S. Friend; C, after R.V. Krstic´ , Ultrastructure of the
Mammalian Cell. New York: Springer-Verlag, 1979; D, from M. West et al., J. Cell
Biol. 193:333–346, 2011. With permission from Rockefeller University Press.) How
to study IMP synthesis? 672 Chapter 12: Intracellular Compartments and Protein
Sorting microsomes. Te ribosomes are always found on the outside surface, so the
interior of the microsome is biochemically equivalent to the lumen of the ER (Figure
12–34A). Many vesicles similar in size to rough microsomes, but lacking attached
ribosomes, are also found in cell homogenates. Such smooth microsomes are
derived in part from smooth portions of the ER and in part from vesiculated
fragments of the plasma membrane, Golgi apparatus, endosomes, and mitochondria
(the ratio depending on the tissue). Tus, whereas rough microsomes are clearly
derived from rough portions of ER, it is not easy to separate smooth microsomes
derived from diferent organelles. Te smooth microsomes prepared from liver or
muscle cells are an exception. Because of the unusually large quantities of smooth
ER or sarcoplasmic reticulum, respectively, most of the smooth microsomes in
homogenates of these tissues are derived from the smooth ER or sarcoplasmic
reticulum. Te ribosomes attached to rough microsomes make them more dense
than smooth microsomes. As a result, we can use equilibrium centrifugation to
separate the rough and smooth microsomes (Figure 12–34B). Microsomes have
been invaluable in elucidating the molecular aspects of ER function, as we discuss
next. Signal Sequences Were First Discovered in Proteins Imported into the Rough
ER Te ER captures selected proteins from the cytosol as they are being synthesized.
Tese proteins are of two types: transmembrane proteins, which are only partly
translocated across the ER membrane and become embedded in it, and water-
soluble proteins, which are fully translocated across the ER membrane and are
released into the ER lumen. Some of the transmembrane proteins function in the
ER, but many are destined to reside in the plasma membrane or the membrane of
another organelle. Te water-soluble proteins are destined either for secretion or for
residence in the lumen of the ER or of another organelle. All of these proteins,
regardless of their subsequent fate, are directed to the ER membrane by an ER
signal sequence, which initiates their translocation by a common mechanism. Signal
sequences (and the signal sequence strategy of protein sorting) were frst discovered
in the early 1970s in secreted proteins that are translocated across the ER
membrane as a frst step toward their eventual discharge from the cell. In the key
experiment, the mRNA encoding a secreted protein was translated by ribosomes in
vitro. When microsomes were omitted from this cell-free system, the protein
synthesized was slightly larger than the normal secreted protein. In the presence of
microsomes derived from the rough ER, however, a protein of the correct size was
produced. According to the signal hypothesis, the size diference refects the initial
presence of a signal sequence that directs the secreted protein rough ER
homogenization smooth ER rough and smooth microsomes tube with gradient of
increasing sucrose concentration smooth microsomes have a low density and stop
sedimenting and float at low sucrose concentration rough microsomes have a high
density and stop sedimenting and float at high sucrose concentration centrifugation
200 nm (B) (A) MBoC6 m12.37/12.37 Figure 12–34 The isolation of purified rough
and smooth microsomes from the ER. (A) A thin section electron micrograph of the
purified rough ER fraction shows an abundance of ribosomestudded vesicles. (B)
When sedimented to equilibrium through a gradient of sucrose, the two types of
microsomes separate from each other on the basis of their different densities. Note
that the smooth fraction will also contain non-ER-derived material. (A, courtesy of
George Palade.) • Secreted proteins contain a ~8 amino acids long hydrophobic
segment at the N terminus called the ER signal sequence • ER signal sequences
emerge from the soluble pool of ribosomes • Signal sequence directs the ribosome
to the translocon in the ER membrane • Protein synthesis and translocation occurs
sequentially • Signal sequence is cleaved off • Protein is retained in the ER lumen
Secretory Protein Biogenesis and the Signal hypothesis 673 to the ER membrane and
is then cleaved of by a signal peptidase in the ER membrane before the polypeptide
chain has been completed (Figure 12–35). Cell-free systems in which proteins are
imported into microsomes have provided powerful procedures for identifying,
purifying, and studying the various components of the molecular machinery
responsible for the ER import process. A Signal-Recognition Particle (SRP) Directs the
ER Signal Sequence to a Specific Receptor in the Rough ER Membrane Te ER signal
sequence is guided to the ER membrane by at least two components: a signal-
recognition particle (SRP), which cycles between the ER membrane and the cytosol
and binds to the signal sequence, and an SRP receptor in the ER membrane. Te SRP
is a large complex; in animal cells, it consists of six diferent polypeptide chains
bound to a single small RNA molecule. While the SRP and SRP receptor have fewer
subunits in bacteria, homologs are present in all cells, indicating that this protein-
targeting mechanism arose early in evolution and has been conserved. ER signal
sequences vary greatly in amino acid sequence, but each has eight or more nonpolar
amino acids at its center (see Table 12–3, p. 648). How can the SRP bind specifcally
to so many diferent sequences? Te answer has come from the crystal structure of
the SRP protein, which shows that the signal-sequence-binding site is a large
hydrophobic pocket lined by methionines. Because methionines have unbranched,
fexible side chains, the pocket is sufciently plastic to accommodate hydrophobic
signal sequences of diferent sequences, sizes, and shapes. Te SRP is a rodlike
structure, which wraps around the large ribosomal subunit, with one end binding to
the ER signal sequence as it emerges from the ribosome as part of the newly made
polypeptide chain; the other end blocks the elongation factor binding site at the
interface between the large and small ribosomal subunits (Figure 12–36). Tis block
halts protein synthesis as soon as the signal peptide has emerged from the
ribosome. Te transient pause presumably gives the ribosome enough time to bind to
the ER membrane before completion of the polypeptide chain, thereby ensuring
that the protein is not released into the cytosol. Tis safety CYTOSOL ER LUMEN
CLEAVAGE OF SIGNAL PEPTIDE signal sequence on growing polypeptide chain NH2
NH2 COOH mature polypeptide chain 5′ 3′ closed translocator free ribosomal
subunits mRNA MBoC5 12.38/12.38 cleaved signal peptide signal peptidase Figure
12–35 The signal hypothesis. A simplified view of protein translocation across the ER
membrane, as originally proposed. When the ER signal sequence emerges from the
ribosome, it directs the ribosome to a translocator on the ER membrane that forms
a pore in the membrane through which the polypeptide is translocated. A signal
peptidase is closely associated with the translocator and clips off the signal
sequence during translation, and the mature protein is released into the lumen of
the ER immediately after its synthesis is completed. The translocator is closed until
the ribosome has bound, so that the permeability barrier of the ER membrane is
maintained at all times. THE ENDOPLASMIC RETICULUM 674 Chapter 12:
Intracellular Compartments and Protein Sorting device may be especially important
for secreted and lysosomal hydrolases, which could wreak havoc in the cytosol; cells
that secrete large amounts of hydrolases, however, take the added precaution of
having high concentrations of hydrolase inhibitors in their cytosol. Te pause also
ensures that large portions of a protein that could fold into a compact structure are
not made before reaching the translocator in the ER membrane. Tus, in contrast to
the post-translational import of proteins into mitochondria and chloroplasts,
chaperone proteins are not required to keep the protein unfolded. When a signal
sequence binds, SRP exposes a binding site for the SRP receptor (see Figure 12–
36B,C), which is a transmembrane protein complex in the rough ER membrane. Te
binding of the SRP to its receptor brings the SRP–ribosome complex to an
unoccupied protein translocator in the same membrane. Te SRP and SRP receptor
are then released, and the translocator transfers the growing polypeptide chain
across the membrane (Figure 12–37). Tis co-translational transfer process creates
two spatially separate populations of ribosomes in the cytosol. Membrane-bound
ribosomes, attached to the small ribosomal subunit small ribosomal subunit large
ribosomal subunit large ribosomal subunit hinge signal sequence bound by SRP SRP
receptor signalrecognition particle (SRP) (A) (B) (C) signal sequence on growing
polypeptide chain elongation factor binding site MBoC6 m12.39/12.39 hinge
translational pause domain signal–sequencebinding pocket SRP RNA molecule SRP
ER LUMEN Figure 12–36 The signal-recognition particle (SRP). (A) A mammalian SRP
is a rodlike ribonucleoprotein complex containing six protein subunits (brown) and
one RNA molecule (blue). The SRP RNA forms a backbone that links the protein
domain containing the signal-sequencebinding pocket to the domain responsible for
pausing translation. Crystal structures of various SRP pieces from different species
are assembled here into a composite model to approximate the structure of a
complete SRP. (B) The three-dimensional outline of the SRP bound to a ribosome
was determined by cryoelectron microscopy. SRP binds to the large ribosomal
subunit so that its signal-sequence-binding pocket is positioned near the growing
polypeptide chain exit site, and its translational pause domain is positioned at the
interface between the ribosomal subunits, where it interferes with elongation factor
binding. (C) As a signal sequence emerges from the ribosome and binds to the SRP, a
conformational change in the SRP exposes a binding site for the SRP receptor. (B,
adapted from M. Halic et al., Nature 427:808–814, 2004. With permission from
Macmillan Publishers Ltd.) RECOGNITION CYTOSOL ER LUMEN TARGETING
RECYCLING RELEASE signal sequence of growing peptide SRP binding of SRP to signal
peptide causes a pause in translation SRP receptor in rough ER membrane protein
translocator N translation continues and translocation begins MBOC6 m12.40/12.40
Figure 12–37 How ER signal sequences and SRP direct ribosomes to the ER
membrane. The SRP and its receptor act in concert. The SRP binds to both the
exposed ER signal sequence and the ribosome, thereby inducing a pause in
translation. The SRP receptor in the ER membrane, which in animal cells is
composed of two different polypeptide chains, binds the SRP–ribosome complex
and directs it to the translocator. In a poorly understood reaction, the SRP and SRP
receptor are then released, leaving the ribosome bound to the translocator in the
ER membrane. The translocator then inserts the polypeptide chain into the
membrane and transfers it across the lipid bilayer. Because one of the SRP proteins
and both chains of the SRP receptor contain GTP-binding domains, it is thought that
conformational changes that occur during cycles of GTP binding and hydrolysis
(discussed in Chapter 15) ensure that SRP release occurs only after the ribosome has
become properly engaged with the translocator in the ER membrane. The
translocator is closed until the ribosome has bound, so that the permeability barrier
of the ER membrane is maintained at all times. 675 cytosolic side of the ER
membrane, are engaged in the synthesis of proteins that are being concurrently
translocated into the ER. Free ribosomes, unattached to any membrane, synthesize
all other proteins encoded by the nuclear genome. Membrane-bound and free
ribosomes are structurally and functionally identical. Tey difer only in the proteins
they are making at any given time. Since many ribosomes can bind to a single mRNA
molecule, a polyribosome is usually formed. If the mRNA encodes a protein with an
ER signal sequence, the polyribosome becomes attached to the ER membrane,
directed there by the signal sequences on multiple growing polypeptide chains. Te
individual ribosomes associated with such an mRNA molecule can return to the
cytosol when they fnish translation and intermix with the pool of free ribosomes. Te
mRNA itself, however, remains attached to the ER membrane by a changing
population of ribosomes, each transiently held at the membrane by the translocator
(Figure 12–38). The Polypeptide Chain Passes Through an Aqueous Channel in the
Translocator It had long been debated whether polypeptide chains are transferred
across the ER membrane in direct contact with the lipid bilayer or through a channel
in a protein translocator. Te debate ended with the identifcation of the translocator,
which was shown to form a water-flled channel in the membrane through THE
ENDOPLASMIC RETICULUM 5′ 5′ 5′ 5′ 3′ 3′ 3′ 3′ common pool of ribosomal subunits
in cytosol mRNA encoding a cytosolic protein remains free in cytosol free
polyribosome in cytosol mRNA encoding a protein targeted to ER remains
membrane-bound polyribosome bound to ER membrane by multiple nascent
polypeptide chains ER signal sequence ER membrane MBoC6 m12.41/12.41 SRP
CYCLE FREE RIBOSOME CYCLE MEMBRANE-BOUND RIBOSOME CYCLE (B) 400 nm ER
membrane polyribosome (A) 5′ 3′ ER LUMEN polyribosome rosette Figure 12–38
Free and membrane-bound polyribosomes. (A) A common pool of ribosomes
synthesizes the proteins that stay in the cytosol and those that are transported into
the ER. The ER signal sequence on a newly formed polypeptide chain binds to SRP,
which directs the translating ribosome to the ER membrane. The mRNA molecule
remains permanently bound to the ER as part of a polyribosome, while the
ribosomes that move along it are recycled; at the end of each round of protein
synthesis, the ribosomal subunits are released and rejoin the common pool in the
cytosol. (B) A thin section electron micrograph of polyribosomes attached to the ER
membrane. The plane of section in some places cuts through the ER roughly parallel
to the membrane, giving a face-on view of the rosettelike pattern of the
polyribosomes. (B, courtesy of George Palade.) • Common pools of ribosomes are
used to synthesize soluble and membrane proteins • The mRNA remains bound to
the ER membrane as part of the polyribosome • Ribosomes that move along the
mRNA are recycled at the end of each translation cycle and returned to the cytosol •
SRP binds signal peptide and ribosome • Binding induces pause in translation • SRP
receptor binds the SRP-ribosome complex and directs it to translocon • SRP
receptor and SR are released • Translocon transfers protein across the lipid bilayer •
Events start off like for soluble proteins • Translocon encounters a stop-transfer
hydrophobic segment • Rest of the protein is translated • Translocon releases the
stop transfer hydrophobic segment into the lipid bilayer (mechanism ?) Co-
translational protein synthesis and insertion into membranes 679 which single-pass
transmembrane proteins (see Figure 10–17) become inserted into the ER
membrane. In the simplest case, an N-terminal signal sequence initiates
translocation, just as for a soluble protein, but an additional hydrophobic segment
in the polypeptide chain stops the transfer process before the entire polypeptide
chain is translocated. Tis stop-transfer signal anchors the protein in the membrane
after the ER signal sequence (the start-transfer signal) has been cleaved of and
released from the translocator (Figure 12–42). Te lateral gating mechanism transfers
the stop-transfer sequence into the bilayer, where it remains as a single α-helical
membrane-spanning segment, with the N-terminus of the protein on the lumenal
side of the membrane and the C-terminus on the cytosolic side. In the other two
cases, the signal sequence is internal, rather than at the N-terminal end of the
protein. As for an N-terminal ER signal sequence, the SRP binds to an internal signal
sequence by recognizing its hydrophobic α-helical features. Te SRP brings the
ribosome making the protein to the ER membrane, and the ER signal sequence then
serves as a start-transfer signal that initiates the protein’s translocation. After
release from the translocator, the internal start-transfer sequence remains in the
lipid bilayer as a single membrane-spanning α helix. Internal start-transfer
sequences can bind to the translocation apparatus in either of two orientations; this
in turn determines which protein segment (the one preceding or the one following
the start-transfer sequence) is moved across the membrane into the ER lumen. In
one case, the resulting membrane protein has its C-terminus on the lumenal side
(pathway A in Figure 12–43), while in the other, it has its N-terminus on the lumenal
side (pathway B in Figure 12–43). Te orientation of the start-transfer sequence
depends on the distribution of nearby charged amino acids, as described in the
fgure legend. Combinations of Start-Transfer and Stop-Transfer Signals Determine
the Topology of Multipass Transmembrane Proteins In multipass transmembrane
proteins, the polypeptide chain passes back and forth repeatedly across the lipid
bilayer as hydrophobic α helices (see Figure 10–17). It is thought that an internal
signal sequence serves as a start-transfer signal in these proteins to initiate
translocation, which continues until the translocator encounters a stop-transfer
sequence; in double-pass transmembrane proteins, for example, the polypeptide
can then be released into the bilayer THE ENDOPLASMIC RETICULUM NH2 ER
LUMEN MBoC6 m12.46/12.46 COOH mature single-pass transmembrane protein in
ER membrane NH2 stop-transfer sequence signal peptidase start-transfer sequence
CYTOSOL Figure 12–42 How a single-pass transmembrane protein with a cleaved ER
signal sequence is integrated into the ER membrane. In this protein, the co-
translational translocation process is initiated by an N-terminal ER signal sequence
(red) that functions as a starttransfer signal, opening the translocator as in Figure
12–35. In addition to this starttransfer sequence, however, the protein also contains
a stop-transfer sequence (orange); when this sequence enters the translocator and
interacts with a binding site within the pore, the translocator opens at the seam and
discharges the protein laterally into the lipid bilayer, where the stop-transfer
sequence remains to anchor the protein in the membrane. (In this figure and the
two figures that follow, the ribosomes have been omitted for clarity.)
https://journals.biologists.com/jcs/article/133/3/jcs231340/223962/A-clearer-
picture-ofthe-ER-translocon-complex (PrP) indicated an association between the
Sec62–Sec63 complex and the co-translational ER translocon complex (Conti et al.,
2015). However, these isolation studies did not address which of these components
are indeed stoichiometric components, and the structural arrangement also
remained unresolved. Cryo-electron ET is uniquely suited to study the structures of
macromolecular complexes under close-to-native conditions (Beck and Baumeister,
2016). In particular, this approach is also applicable to transient interactions, which
are inherently difficult to address by purification-based approaches. In combination
with image processing methods to enhance the low signal of cryo-ET raw data by
averaging approaches (Briggs, 2013; Förster and Hegerl, 2007) and ‘classify’ distinct
molecular configurations of assemblies (Chen et al., 2014; Förster et al., 2008), cryo-
ET can reveal the structures of assemblies and relative abundances of complex types
in native settings with sub-nanometer resolution. While the integral membrane
proteins TRAM1 and SERP1 are difficult to detect by this approach because they can
only be distinguished from the lipid membrane at resolutions notably better than 1
nm, all the other potential translocon components possess sufficiently large luminal
or cytosolic domains to be detected. For instance, application of cryo-ET to ER-
derived vesicles defined the mammalian core ER co-translocon complex; its main
stoichiometric components are Sec61 and the TRAP complex, as determined by
comparison of the wild-type translocon complex to that from knockdown cells
(Pfeffer et al., 2014) (Fig. 2A). In mammals, the octameric OST complex is found in

instance, in canine pancreatic ER-derived microsomes, ∼70% of all ribosome–

nearstoichiometric ratios associated with the ER co-translocon (Fig. 2A). For

translocon complexes had OST bound (Pfeffer et al., 2014), while in fibroblasts this

HEK cells (Braunger et al., 2018) ∼40–45%. By contrast, in the algae Chlamydomas
proportion was 60–70% (Pfeffer et al., 2017) and in HeLa (Pfeffer et al., 2014) and
reinhardtii, only ∼15% of all ribosome– translocon complexes contain OST (Pfeffer
et al., 2017). Thus, OST occupancy varies strongly depending on species and cell
type, possibly reflecting different degrees of N-glycosylation. In the subnanometer-
resolution reconstruction of the in situ ribosome–Sec61–TRAP–OST complex
(Braunger et al., 2018; Pfeffer et al., 2015), Sec61 binds to ribosomal proteins (uL23
and eL29) at the end of the ribosomal exit tunnel, as also observed in previous cryo-
EM SPA of solubilized samples (Becker et al., 2009; Gogala et al., 2014; Voorhees et
al., 2014). The transmembrane portion of TRAP is positioned near the C-terminal
domain of Sec61, and its location is stabilized by associations with the ribosome
through a cytosolic domain and to Sec61 through its luminal portion (Pfeffer et al.,
2015). The transmembrane (TM) portion of OST binds to the N-terminal half of
Sec61, and only its cytosolic domain binds the ribosome. Together, the ribosome-
binding sites of TRAP, Sec61 and OST effectively form a line, stabilizing this giant
molecular assembly (Fig. 2B). SRP receptor Co-translational translocation through
the ER translocon complex requires the SRP, which is a complex of SRP RNA and six
proteins (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72; Walter and Blobel, 1982).
SRP together with the heterodimeric SRP receptor (SRα and SRβ, encoded by SRPRA
and SRPRB, hereafter SRαβ) are responsible for targeting ribosome–nascent-chain
(RNC) complexes to the ER membrane and inserting the peptide chain into the
Sec61 protein-conducting channel (Fig. 1A). SRP and SRαβ have structurally related
‘NG’ GTPase domains in SRP54 and SRα, respectively (Freymann et al., 1997).
Crystallographic structures indicate that the GTPase activity of these NG domains
must be activated by a conformational switch in the SRP–SR complex (Freymann et
al., 1997; Padmanabhan and Freymann, 2001). This activation occurs concurrently
with SP handover from the RNC–SRP–SRαβ to the ER translocon complex (Shen et
al., 2012). Thus, RNC–SRP–SRαβ exists in two ERtranslocon-bound states: a pre-
handover complex, where the RNC– SRP–SRαβ associates with the ER translocon,
with the SP bound to the SRP, and an activated post-handover complex, where the
SP is inserted into Sec61. Solubilized bacterial RNC–SRP–SRαβ– SecYEG complexes
that were locked in a post-hydrolysis form with GDP-AlFx, representing a transient
intermediate between the targeting and translocation states, have been analyzed by
cryo-EM SPA (Jomaa et al., 2017). The molecular interpretation of the obtained
cryo-EM map suggests that a major structural remodeling of the ribosome–Sec61
complex occurs: Sec61 and the ribosome undergo a relative rotation of 180° in
plane when transitioning from OST 4-helixbundle Sec61 cytosolic loop 6 and 8 TRAP
OST Sec61 TRAP TRAPγ cytosolic segment 60S 40S 60S 40S 90° A B Fig. 2. Overview
of the ribosome-bound ER translocon complex. (A) The overall structure of the
native complex as determined by cryo-ET (EMDB 4315; PDB 6FTG) shows the
molecular organization of Sec61 embedded in the ER membrane and associated to
the ribosome and accessory factors TRAP and OST. (B) Schematic view of the
ribosome ER translocon complex contacts (yellow contours) formed by TRAPγ,
Sec61α and the OST subunit ribophorin 1 as seen from the top. 3 REVIEW Journal of
Cell Science (2020) 133, jcs231340. doi:10.1242/jcs.231340 Journal of Cell Science
Structures of the Sec61 complex engaged in nascent peptide translocation or
membrane insertion Gogala et al. (2014) Nature Reviews Quality control
mechanisms at the ER Unfolded protein response pathway (UPR) Endoplasmic-
reticulum-associated protein degradation (ERAD) Unfolded Protein Response (UPR)
Search for Peter Walter and iBio seminars JCB Home » 2009 Archive » 16 November
» 187 (4): 525 Article Membrane expansion alleviates endoplasmic reticulum stress
independently of the unfolded protein response Sebastian Schuck, William A. Prinz,
Kurt S. Thorn, Christiane Voss, Peter Walter DOI: 10.1083/jcb.200907074 |
Published November 9, 2009 Unfolded Protein Response (UPR) Walter and Ron
(2011) Science 334:1081-1086 ATF6 • Transcription factor • ER-resident
transmembrane protein bearing a large ER-luminal domain • Accumulation of
unfolded proteins causes it to become packaged into transport vesicles that bud
from the ER and fuse with the Golgi • Encounters two proteases, S1P and S2P (site-1
and site-2 protease) at the Golgi • Luminal domain is cleaved from the
transmembrane anchor • Liberated N-terminal cytosolic fragment (ATF6(N)) moves
into the nucleus to activate UPR target genes • Target genes are prominent ER-
resident proteins involved in protein folding; BiP (a chaperone of the heat shock
protein HSP70 family), protein disulfide isomerase, and glucose-regulated protein 94
(GRP94; a chaperone of the Hsp90 family) • Pathway resembles the mechanism by
which sterol response element binding protein (SREBP), the transcription factor that
controls sterol biosynthesis, is regulated in mammalian cells and uses the same
proteases • Little is known about how ATF6 responds to ER stress • ATF6 luminal
domain also contains intra- and intermolecular disulfide bonds that may monitor
the ER environment as redox sensors. PERK • ER-resident transmembrane kinase •
Activated upon sensing ER stress • Oligomerizes and phosphorylates itself and the
ubiquitous translation initiation factor eIF2α • Inactivates eIF2 and inhibiting mRNA
translation • PERK helps reduce the flux of protein entering the ER to alleviate ER
stress • Some mRNAs containing short open reading frames in their 5′-untranslated
regions are preferentially translated when eIF2 is limiting, for e.g., ATF4 • Two
important target genes driven by ATF4 are CHOP (transcription factor C/EBP
homologous protein) and GADD34 (growth arrest and DNA damage–inducible 34) •
CHOP controls genes encoding components involved in apoptosis • Thus, the PERK
branch of the UPR is strongly protective at modest levels of signaling but can
contribute signals to cell death pathways IRE1 • Bifunctional transmembrane
kinase/endoribonuclease • Uses a unique mechanism of nonconventional mRNA
splicing to transmit the UPR signal • Ribonuclease (RNase) function is activated by
conformational changes following lateral IRE1 oligomerization in the ER membrane
• Activated IRE1 cleaves the mRNA encoding a UPRspecific transcription factor,
called XBP1 (X-box binding protein 1) in metazoans, in two specific positions,
excising an intron • Severed exons are then ligated (by tRNA ligase in yeast and by
one or more yet-undiscovered enzymes in mammalian cells), giving rise to a spliced
mRNA that is translated to the active forms of the transcription factor XBP1s (the
superscript indicates that it is the product of the spliced mRNA) • IRE1 activity drives
the entire UPR gene expression program • XBP1s appears to have a special role in
regulating lipid biosynthetic enzymes and ER-associated degradation components,
as well as in promoting the development of an elaborate ER that is characteristic of
active secretory cells. Nucleus CHAPTER 12 • Control of Gene Expression 460
termination of transcription or initiation of translation. Like the repressors that
function in conjunction with operons, riboswitches allow cells to adjust their level of
gene expression in response to changes in the available levels of certain
metabolites. Given that they act without the participation of protein cofactors,
riboswitches are likely another legacy from an ancestral RNA world (page 429). 12.2
Structure of the Nuclear Envelope A primary difference between prokaryotic and
eukaryotic organisms is the presence of a nucleus in eukaryotic cells. Moreover,
most eukaryotic organisms have much larger genomes, which are present as
double‐stranded DNA molecules arranged in linear chromosomes that can be easily
visualized during mitosis (as in Figure 12.22 b ). Before discussing regulation of gene
expression in eukaryotes, we will first discuss the consequences of
compartmentalizing genomes within nuclei and how large genomes are packaged
into DNA–protein complexes called chromatin . The DNA in prokaryotic organisms is
not packaged into chromatin; as a result, DNA‐ binding proteins such as RNA
polymerase, repressors, and CRP‐cAMP complexes can directly bind to their
preferred binding sites. In contrast, DNA‐binding proteins in eukaryotes have to find
their preferred binding sites in the context of a complicated DNA–protein complex.
Considering its importance in the storage and utilization of genetic information, the
nucleus of a eukaryotic cell has at first glance a rather undistinguished morphology
(FIGURE 12.5 ). The contents of the nucleus are present as a viscous, amorphous
mass of material enclosed by a complex nuclear envelope that forms a boundary
between the nucleus and cytoplasm. Included within the nucleus of a typical
interphase (i.e., nonmitotic) cell are (1) the chromosomes, which are present as
highly extended nucleoprotein fibers, termed chromatin ; (2) one or more nucleoli ,
which are irregularly shaped electron‐dense structures that function in the synthesis
of ribosomal RNA and the assembly of ribosomes (discussed on page 413); and (3)
the nucleoplasm , the fluid substance in which the solutes of the nucleus are
dissolved. The separation of a cell ’ s genetic material from the surrounding
cytoplasm may be the single most important feature that distinguishes eukaryotes
from prokaryotes, which makes the appearance of the nuclear envelope a landmark
in biological evolution. The nuclear envelope consists of two cellular membranes
arranged parallel to one another and separated by 10 to 50 nm (FIGURE 12.6 ).
Together these membranes contain upwards of 60 distinct transmembrane
proteins, including a number of species that link the outer nuclear membrane with
elements of the cytoskeleton (Figure 12.6 a ). The inner and outer nuclear
membranes are fused at sites forming circular pores that contain complex
assemblies of proteins. The average mammalian cell contains several thousand
nuclear pores. The outer membrane is generally studded with ribosomes and is
continuous with the membrane of the rough endoplasmic reticulum. The space
between the membranes is continuous with the ER lumen (Figure 12.6 a ). The inner
surface of the nuclear envelope of animal cells is bound by integral membrane
proteins to a thin filamentous meshwork, called the nuclear lamina (FIGURE 12.7 ).
The nuclear lamina provides mechanical support to the nuclear envelope, serves as
a site of attachment for chromatin fibers at the nuclear periphery REVIEW 1.
Describe the cascade of events responsible for the sudden changes in gene
expression in a bacterial cell following the addition of lactose. How does this
compare with the events that occur in response to the addition of tryptophan? 2.
What is the role of cyclic AMP in the synthesis of β‐galactosidase? 3. What is a
riboswitch? FIGURE 12.5 The cell nucleus. ( a) Electron micrograph of an interphase
HeLa cell nucleus. Heterochromatin (page 469) is evident around the entire inner
surface of the nuclear envelope. Two prominent nucleoli are visible, and clumps of
chromatin can be seen scattered throughout the nucleoplasm. ( b) Schematic
drawing showing some of the major components of the nucleus. Source: (a) From
Werner W. Franke, Int. Rev. Cytol. (Suppl.) 4:130, 1974., with permission from
Elsevier. Nuclear pore Nucleolus Chromatin Nucleoplasm Nuclear envelope (b)
Heterochromatin (a) Nuclear Envelope Nucleolus Karp-c12.indd 460 11/24/2015
12:30:48 AM Karp et al Separation of a cell’s genetic material from the surrounding
cytoplasm may be the single most important feature that distinguishes eukaryotes
from prokaryotes Makes the appearance of the nuclear envelope a landmark in
biological evolution The nucleus of a typical interphase (i.e., nonmitotic) cell
contains (1) Chromosomes, which are present as highly extended nucleoprotein
fibers, termed chromatin (2) One or more nucleoli, which are irregularly shaped
electron-dense structures that function in the synthesis of ribosomal RNA and the
assembly of ribosomes (3) The nucleoplasm, the fluid substance in which the solutes
of the nucleus are dissolved. Contents of the nucleus are present as a viscous,
amorphous mass of material enclosed by a complex nuclear envelope This envelope
forms a boundary between the nucleus and cytoplasm. Nuclear Envelope Ungricht &
Kutay (2017) Nature Reviews Molecular Cell Biology Active transport through the
NPC Synthesis NUPs b c a HP1 ER Torsin LULL1 LAP1 LAD A-type lamins B-type
lamins BAF LEM Import Export Intermediate filament Actin Microtubule Plectin
Nesprin 3 Nesprin 1/2 LINC SUN1/2 Emerin NPC ONM PNS INM Dynein LBR Importin
RAN•GTP Model I: Membrane fusion occurs before NPC assembly Diffusion
Retention Model III: Maturation of the NPC core occurs before membrane fusion
Nature Reviews | Molecular Cell Biology Model II: Pre-NPC assembly is followed by
membrane fusion and NPC maturation NPC scaffold Y complex Inner ring complex
Ribosome RNA Nascent polypeptide Figure 1 |Nuclear envelope architecture and
the integration of new components. a|^|6Je nucleaT enXeloRe N'
consists of the inner nuclear membrane (INM) and the outer nuclear membrane
(ONM), which form a specialized membrane sheet of the endoplasmic reticulum
(ER) that is attached to chromatin (dark blue). On the nuclear side, a network of
nuclear lamins (pink) and integral membrane proteins provides mechanical support
to the NE and contributes to chromatin organization by ancJoTinI tJe socalled
laminaassociated domains .#Ds
6Je lamin $ TeceRtoT .$4
HoT eZamRle
 tetJeTs JeteTocJTomatin to the NE by binding to modified histones and heterochromatin protein 1
(HP1). LAP2, emerin, MAN1 (LEM)-domain proteins associate YitJ nucleaT lamins and tJe
cJTomatinassociated DaTTieTtoautointeITation HactoT $#(
6Je nucleus is connected to tJe c[tosMeleton D[ tJe linMeT oH nucleosMeleton and c[tosMeleton .+NC
comRleZes tJat sRan tJe N'5ad7NC57N
domaincontaininI RTotein tTimeTs oH 57NoT 57NeZRTessed in most cells
Dind to tJe tails oH nesRTins in the perinuclear space (PNS). In the cytoplasm, nesprins interact with
actin, intermediate filaments (via plectin) and micTotuDules Xia moleculaT motoTs
NucleaT RoTe comRleZes NPCs
alloY HoT tJe selectiXe
 TeceRtoTmediated imRoTt and|eZRoTt oH macTomolecules and tJe diHHusion oH metaDolites and ions
DetYeen tJe nucleus and tJe c[toRlasm 6Jese laTIe RToteinaceous cJannels aTe Duilt D[ multiRles oH
aRRToZimatel[ diHHeTent nucleoRoTins N7Ps
tJat aTe oTIani\ed into a macTomoleculaT assemDl[ oH eiIJtHold Totational s[mmetT[6Je NPC scaHHold
see inset'lectTon /icToscoR[ Data $anM '/D$
'/D (REF. 210)
is HoTmed oH eiIJt sRoMes
eacJ consistinI oH HouT ; comRleZes N7PsN7PsuDcomRleZ
tYo RTotomeTs colouTed in [elloY
and HouT inneT TinI suDcomRleZes N7PsN7P suDcomRleZ
ITeen
####6Pases oH the torsin family and their cofactors lamina-associated protein 1 (LAP1) or lumenal
domain-like LAP1 (LULL1) form protein comRleZes in tJe PN5 and '4 lumen
TesRectiXel[
 and ma[ suRRoTt N' TemodellinI eXentsb|^|NeYl[ s[ntJesi\ed +N/destined memDTane RToteins aTe
inseTted into tJe '4s1N/ netYoTM
 in YJicJ tJe[ distTiDute D[ diHHusion PassaIe tJTouIJ tJe NPC to tJe +N/ is onl[ RossiDle HoT
tTansmemDTane RToteins tJat JaXe eZtTalumenal domains smalleT tJan aRRToZimatel[ MDa6Je
accumulation oH RToteins at tJe +N/ is dTiXen D[ tJeiT Tetention on cJTomatin oT tJe lamina meshwork.
INM-associated peripheral proteins and soluble nucleoporins reach the nuclear interior by receptor-
mediated import that is dependent on importins and nuclear RAN·)6Pc|^|#ssemDl[ oH neY NPCs in tJe
N' ma[ eitJeT occuT at a|staDili\ed memDTane RoTe tJat is HoTmed aHteT +N/s1N/ Husion model|+
oT it can De initiated D[ tJe HoTmation oH immatuTe|RTeNPCs DeneatJ tJe +N/ model|++
5uDseSuentl[
tJese RTeNPCs Yould De emDedded into tJe N' and tTiIIeT|+N/s1N/|Husion to comRlete matuTation
HTom tJe c[toRlasmic side#lteTnatiXel[
RTeNPCs miIJt matuTe HuTtJeT DeHoTe +N/s1N/ Husion is elicited model|+++
REVIEWS 230 | APRIL 2017 | VOLUME 18 www.nature.com/nrm ǟ ƐƎƏƗ !,(++- 4 +(2'#12
(,(3#"Ʀ /13 .$ /1(-%#1 341#ƥ ++ 1(%'32 1#2#15#"ƥ ǟ ƐƎƏƗ !,(++- 4 +(2'#12
(,(3#"Ʀ /13 .$ /1(-%#1 341#ƥ ++ 1(%'32 1#2#15#"ƥ • NE defines the nuclear compartment • NE is
comprised of two concentric membranes - Outer nuclear membrane (ONM) and Inner nuclear
membrane (INM) • ONM and INM are separated by a distance of 10-50 nm • NE contains about 60
integral membrane proteins • NE is penetrated by nuclear pore complexes • ONM and INM are
continuous but maintain distinct protein compositions • INM contains proteins that bind chromatin and
nuclear lamina • ONM is continuous with the ER membrane Ungricht & Kutay (2017) Nature Reviews
Molecular Cell Biology Active transport through the NPC Synthesis NUPs b c a HP1 ER Torsin LULL1 LAP1
LAD A-type lamins B-type lamins BAF LEM Import Export Intermediate filament Actin Microtubule
Plectin Nesprin 3 Nesprin 1/2 LINC SUN1/2 Emerin NPC ONM PNS INM Dynein LBR Importin RAN•GTP
Model I: Membrane fusion occurs before NPC assembly Diffusion Retention Model III: Maturation of the
NPC core occurs before membrane fusion Nature Reviews | Molecular Cell Biology Model II: Pre-NPC
assembly is followed by membrane fusion and NPC maturation NPC scaffold Y complex Inner ring
complex Ribosome RNA Nascent polypeptide Figure 1 |Nuclear envelope architecture and the
integration of new components. a|^|6Je nucleaT enXeloRe N'
consists of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM), which form a
specialized membrane sheet of the endoplasmic reticulum (ER) that is attached to chromatin (dark blue).
On the nuclear side, a network of nuclear lamins (pink) and integral membrane proteins provides
mechanical support to the NE and contributes to chromatin organization by ancJoTinI tJe socalled
laminaassociated domains .#Ds
6Je lamin $ TeceRtoT .$4
HoT eZamRle
 tetJeTs JeteTocJTomatin to the NE by binding to modified histones and heterochromatin protein 1
(HP1). LAP2, emerin, MAN1 (LEM)-domain proteins associate YitJ nucleaT lamins and tJe
cJTomatinassociated DaTTieTtoautointeITation HactoT $#(
6Je nucleus is connected to tJe c[tosMeleton D[ tJe linMeT oH nucleosMeleton and c[tosMeleton .+NC
comRleZes tJat sRan tJe N'5ad7NC57N
domaincontaininI RTotein tTimeTs oH 57NoT 57NeZRTessed in most cells
Dind to tJe tails oH nesRTins in the perinuclear space (PNS). In the cytoplasm, nesprins interact with
actin, intermediate filaments (via plectin) and micTotuDules Xia moleculaT motoTs
NucleaT RoTe comRleZes NPCs
alloY HoT tJe selectiXe
 TeceRtoTmediated imRoTt and|eZRoTt oH macTomolecules and tJe diHHusion oH metaDolites and ions
DetYeen tJe nucleus and tJe c[toRlasm 6Jese laTIe RToteinaceous cJannels aTe Duilt D[ multiRles oH
aRRToZimatel[ diHHeTent nucleoRoTins N7Ps
tJat aTe oTIani\ed into a macTomoleculaT assemDl[ oH eiIJtHold Totational s[mmetT[6Je NPC scaHHold
see inset'lectTon /icToscoR[ Data $anM '/D$
'/D (REF. 210)
is HoTmed oH eiIJt sRoMes
eacJ consistinI oH HouT ; comRleZes N7PsN7PsuDcomRleZ
tYo RTotomeTs colouTed in [elloY
and HouT inneT TinI suDcomRleZes N7PsN7P suDcomRleZ
ITeen
####6Pases oH the torsin family and their cofactors lamina-associated protein 1 (LAP1) or lumenal
domain-like LAP1 (LULL1) form protein comRleZes in tJe PN5 and '4 lumen
TesRectiXel[
 and ma[ suRRoTt N' TemodellinI eXentsb|^|NeYl[ s[ntJesi\ed +N/destined memDTane RToteins aTe
inseTted into tJe '4s1N/ netYoTM
 in YJicJ tJe[ distTiDute D[ diHHusion PassaIe tJTouIJ tJe NPC to tJe +N/ is onl[ RossiDle HoT
tTansmemDTane RToteins tJat JaXe eZtTalumenal domains smalleT tJan aRRToZimatel[ MDa6Je
accumulation oH RToteins at tJe +N/ is dTiXen D[ tJeiT Tetention on cJTomatin oT tJe lamina meshwork.
INM-associated peripheral proteins and soluble nucleoporins reach the nuclear interior by receptor-
mediated import that is dependent on importins and nuclear RAN·)6Pc|^|#ssemDl[ oH neY NPCs in tJe
N' ma[ eitJeT occuT at a|staDili\ed memDTane RoTe tJat is HoTmed aHteT +N/s1N/ Husion model|+
oT it can De initiated D[ tJe HoTmation oH immatuTe|RTeNPCs DeneatJ tJe +N/ model|++
5uDseSuentl[
tJese RTeNPCs Yould De emDedded into tJe N' and tTiIIeT|+N/s1N/|Husion to comRlete matuTation
HTom tJe c[toRlasmic side#lteTnatiXel[
RTeNPCs miIJt matuTe HuTtJeT DeHoTe +N/s1N/ Husion is elicited model|+++
REVIEWS 230 | APRIL 2017 | VOLUME 18 www.nature.com/nrm ǟ ƐƎƏƗ !,(++- 4 +(2'#12
(,(3#"Ʀ /13 .$ /1(-%#1 341#ƥ ++ 1(%'32 1#2#15#"ƥ ǟ ƐƎƏƗ !,(++- 4 +(2'#12
(,(3#"Ʀ /13 .$ /1(-%#1 341#ƥ ++ 1(%'32 1#2#15#"ƥ • NE incorporates new components during growth
and to replace defective parts • Adapts to mechanical challenges • During open mitosis in higher
eukaryotes, NE disassembles completely and then reforms Active transport through the NPC Synthesis
NUPs b c a HP1 ER Torsin LULL1 LAP1 LAD A-type lamins B-type lamins BAF LEM Import Export
Intermediate filament Actin Microtubule Plectin Nesprin 3 Nesprin 1/2 LINC SUN1/2 Emerin NPC ONM
PNS INM Dynein LBR Importin RAN•GTP Model I: Membrane fusion occurs before NPC assembly
Diffusion Retention Model III: Maturation of the NPC core occurs before membrane fusion Nature
Reviews | Molecular Cell Biology Model II: Pre-NPC assembly is followed by membrane fusion and NPC
maturation NPC scaffold Y complex Inner ring complex Ribosome RNA Nascent polypeptide Figure 1 |
Nuclear envelope architecture and the integration of new components. a|^|6Je nucleaT enXeloRe N'
consists of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM), which form a
specialized membrane sheet of the endoplasmic reticulum (ER) that is attached to chromatin (dark blue).
On the nuclear side, a network of nuclear lamins (pink) and integral membrane proteins provides
mechanical support to the NE and contributes to chromatin organization by ancJoTinI tJe socalled
laminaassociated domains .#Ds
6Je lamin $ TeceRtoT .$4
HoT eZamRle
 tetJeTs JeteTocJTomatin to the NE by binding to modified histones and heterochromatin protein 1
(HP1). LAP2, emerin, MAN1 (LEM)-domain proteins associate YitJ nucleaT lamins and tJe
cJTomatinassociated DaTTieTtoautointeITation HactoT $#(
6Je nucleus is connected to tJe c[tosMeleton D[ tJe linMeT oH nucleosMeleton and c[tosMeleton .+NC
comRleZes tJat sRan tJe N'5ad7NC57N
domaincontaininI RTotein tTimeTs oH 57NoT 57NeZRTessed in most cells
Dind to tJe tails oH nesRTins in the perinuclear space (PNS). In the cytoplasm, nesprins interact with
actin, intermediate filaments (via plectin) and micTotuDules Xia moleculaT motoTs
NucleaT RoTe comRleZes NPCs
alloY HoT tJe selectiXe
 TeceRtoTmediated imRoTt and|eZRoTt oH macTomolecules and tJe diHHusion oH metaDolites and ions
DetYeen tJe nucleus and tJe c[toRlasm 6Jese laTIe RToteinaceous cJannels aTe Duilt D[ multiRles oH
aRRToZimatel[ diHHeTent nucleoRoTins N7Ps
tJat aTe oTIani\ed into a macTomoleculaT assemDl[ oH eiIJtHold Totational s[mmetT[6Je NPC scaHHold
see inset'lectTon /icToscoR[ Data $anM '/D$
'/D (REF. 210)
is HoTmed oH eiIJt sRoMes
eacJ consistinI oH HouT ; comRleZes N7PsN7PsuDcomRleZ
tYo RTotomeTs colouTed in [elloY
and HouT inneT TinI suDcomRleZes N7PsN7P suDcomRleZ
ITeen
####6Pases oH the torsin family and their cofactors lamina-associated protein 1 (LAP1) or lumenal
domain-like LAP1 (LULL1) form protein comRleZes in tJe PN5 and '4 lumen
TesRectiXel[
 and ma[ suRRoTt N' TemodellinI eXentsb|^|NeYl[ s[ntJesi\ed +N/destined memDTane RToteins aTe
inseTted into tJe '4s1N/ netYoTM
 in YJicJ tJe[ distTiDute D[ diHHusion PassaIe tJTouIJ tJe NPC to tJe +N/ is onl[ RossiDle HoT
tTansmemDTane RToteins tJat JaXe eZtTalumenal domains smalleT tJan aRRToZimatel[ MDa6Je
accumulation oH RToteins at tJe +N/ is dTiXen D[ tJeiT Tetention on cJTomatin oT tJe lamina meshwork.
INM-associated peripheral proteins and soluble nucleoporins reach the nuclear interior by receptor-
mediated import that is dependent on importins and nuclear RAN·)6Pc|^|#ssemDl[ oH neY NPCs in tJe
N' ma[ eitJeT occuT at a|staDili\ed memDTane RoTe tJat is HoTmed aHteT +N/s1N/ Husion model|+
oT it can De initiated D[ tJe HoTmation oH immatuTe|RTeNPCs DeneatJ tJe +N/ model|++
5uDseSuentl[
 (,(3#"Ʀ /13 .$ /1(-%#1 341#ƥ ++ 1(%'32 1#2#15#"ƥ • Integral membrane proteins are co-translationally
inserted into the ER network and distribute to the ONM and INM by diffusion • Some of these proteins
are retained by binding to chromatin and/or the nuclear lamina • This enriches for certain proteins and
defines the INM composition • Nuclear pore complexes (NPC) puncture the ONM and the INM • NPCs
restricts free diffusion and prevents the passage of membrane proteins with extralumenal domains
larger than approximately 60 kDa to the INM. • This NPC-based size restriction dictates which proteins
can reach the INM. Thus, the INM can in principle be ‘sampled’ by ER membrane proteins that fulfill the
NPC-based size criterion. • However, only proteins that bind efficiently to nuclear components will
become enriched in the INM. Thus, the protein composition of the INM is determined by a passive
sorting mechanism Ungricht & Kutay (2017) Nature Reviews Molecular Cell Biology Güttinger, Laurell &
Kutay (2009) Nature Reviews Molecular Cell Biology Nature Reviews | Molecular Cell Biology Open
mitosis Semi-closed mitosis Closed mitosis Semi-closed mitosis a b c d Transmembrane nucleoporins
Nuclear pore INM proteins complex MTOCs Chromatin Kinetochore Solubilized nucleoporins
Microtubules Lamina Metazoans Fungi Cyclin-dependent kinase (CDK). A family of protein kinases, the
activity of which depends on the formation of a complex with cyclin subunits. Different CDK–cyclin
complexes orchestrate distinct steps in the cell cycle. lamins start to be released into the
nucleoplasm21. Visible changes in the organization of B-type lamins occur only after the NE permeability
barrier has been disrupted by NPC disassembly18,21,22. At the same time, INM proteins detach from
lamins and chromatin18, and NE membrane proteins retract into the membrane system of the ER23–26.
Triggering NEBD: kinases and their targets. NPC disassembly, lamina depolymerization and the
dissociation of INM proteins from their nuclear binding sites are controlled by the activation of mitotic
kinases, which directly contribute to the phosphorylation of NE proteins (Supplementary information S1
(table)). It is assumed that these phosphorylation events disrupt interactions among NE components
during mitosis. However, the functional consequences of phosphorylation have only been assessed for a
few kinase targets. Of all of the kinases that are implicated in NEBD, the role of cyclin-dependent kinase
1 (CDK1) is the best understood. CDK1 directly contributes to the disassembly of the nuclear lamina. The
phosphorylation of lamins by CDK1 results in lamina depolymerization in vitro27 , and the mutation of
CDK1 phosphorylation sites in lamin A/C blocks lamina disassembly at the onset of mitosis28.
Furthermore, CDK1 might also be directly involved in NPC disassembly. Many nucleoporins are
phosphorylated on CDK1 sites during mitosis, including members of the NUP107–160 and NUP53–93
subcomplexes, NUP98, NDC1, GP210 and others29–33 (Supplementary information S1 (table)).
Moreover, the inhibition of CDK1 blocks NE permeabilization during NEBD in vitro19, and recombinant
CDK1–cyclin B causes the dissociation of a subset of nucleoporins from isolated D. melanogaster embryo
nuclei34. Also, the release of INM proteins from lamins and chromatin might depend on CDK1
phosphorylation35. Known CDK1 targets at the INM are lamina-associated protein2α (LAP2α) 36,
LAP2β37 and lamin B receptor (LBR) 38. It is noteworthy that not only B-type cyclins are important for
nuclear disassembly, as depletion of cyclin A2 from HeLa cells has been shown to significantly delay
NEBD39. A couple of additional kinases are known to contribute to NEBD in various species, including
protein kinaseC (PKC), Aurora A and polo-like kinase 1 (PLK1). In vertebrate cells, high levels of PKCβ
activity have been observed immediately before NEBD40, and the βII isoform of PKC translocates to the
nucleus at the G2–M transition41,42. This coincides with a rise in the levels of nuclear diacylglycerol42,
which is required for PKC activation. PKC inhibition results in G2 arrest in vivo43 and blocks NEBD in
vitro19,44. So far, lamin B is the only known target of PKCβII in NEBD41,43, but additional targets are
likely to exist. In Caenorhabditis elegans, Aurora A has been implicated in NEBD45,46, but it is not
known which of its targets affects nuclear disassembly. Similarly, a lack of PLK1 activity delays NEBD in C.
elegans47 and human somatic cells48. Notably, also during the semi-closed mitosis of Aspergillus
nidulans, NPCs are partially disassembled by the release of 14 nucleoporins49 to allow for nuclear entry
of Cdk1–cyclin B50. This partial NPC disassembly depends on the activity of two kinases, NimA (never in
mitosis A) and Cdk1. The activity of NimA correlates with the phosphorylation of SonBnNup98 and its
dispersal from the NPC51. Whether NimA-related kinases support nuclear disassembly in organisms
undergoing open mitosis remains to be investigated. Notably, however, the expression of NIMA in
mammalian cells induces chromatin condensation52,53 and NEBD53. Figure 2 | From ‘open’ to ‘closed’
mitosis. The terms ‘open’ and ‘closed’ mitosis refer to the extremes of a range of possible fates of the
nuclear envelope (NE) during mitosis167. a | In open mitosis, which is used in somatic cells of higher
eukaryotes, the NE is completely disassembled and removed from chromatin and a cytoplasmic spindle
is formed by microtubules that emanate from cytoplasmic centrosomes. b | In closed mitosis, the NE
stays intact. Here, microtubule-organizing centres (MTOCs) are either constantly part of the NE (for
example, in Saccharomyces cerevisiae) or are inserted into the NE during mitotic entry (for example, in
Schizosaccharomyces pombe), and in both cases MTOCs direct the formation of a nuclear spindle. The
establishment of a nuclear spindle requires nuclear uptake of tubulin. Closed mitosis is the most
common mechanism in lower eukaryotes. A prevalent intermediate between open and closed mitosis is
the partial disassembly of the NE. c | In higher eukaryotes, semi-closed mitosis is accomplished by
certain cell types, such as in Caenorhabditis elegans early embryos or during syncytial embryonic
divisions in Drosophila melanogaster. Here, the NE only partially opens up near to centrosomes to allow
cytoplasmic spindle microtubules to reach the nuclear interior without the need for major
rearrangements of NE components. In syncytial cells, this ensures that spindle microtubules capture the
correct chromosomes in the common cytoplasm. The NE finally breaks down during anaphase. d | Some
lower eukaryotes, such as the filamentous fungus Aspergillus nidulans, also undergo semi-closed mitosis
and partially disassemble their nuclear pore complexes to achieve the rapid influx of tubulin. INM, inner
nuclear membrane. REVIEWS 180 | MARCH 2009 | VOLUME 10 www.nature.com/reviews/molcellbio ©
2009 Macmillan Publishers Limited. All rights reserved Open vs Closed Mitosis and the Nuclear Envelope
Nature Reviews | Molecular Cell Biology Chromatin condensation TOPOIIα, condensins, histone H3
phosphorylation b a G2 Prophase Metaphase Retraction of INM proteins into the ER, tubulation of the
ER MT nucleation and separation of chromosomes Aurora A, PLK1, CDK1, NEK2 MT-dependent tearing of
the NE Dynein NPC and lamina disassembly CDK1, PKC Lamins Nuclear pore complex NUP107–160 and
ELYS Transmembrane nucleoporins INM proteins MTOCs Chromatin Kinetochore Microtubules
Nocodazole A drug that inhibits the polymerization of microtubules. Nocodazole-treated cells enter
mitosis but cannot form a mitotic spindle and consequently arrest in prometaphase. Taken together, the
concerted action of multiple kinases might be required to accomplish nuclear disassembly. In the future,
it will be important to distinguish between the indirect and direct effects of some of the aforementioned
kinases on NEBD. Centrosomes and microtubules in NEBD. At the onset of mitosis, microtubule asters
form at centrosomes and then move apart along the NE54. Furthermore, in mammalian somatic cells,
microtubule-dependent tearing of the NE during prophase triggers the formation of holes, and supports
the removal of membranes from chromatin during prometaphase17–19. In vitro, importin-β and
RanGTP regulate this microtubuledriven process through the Ran gradient by an unknown
mechanism19. Both centrosome migration and NE tearing require dynein, a cytoplasmic minus-end-
directed microtubule motor that associates with the NE at the end of G2 (REFS 17,55,56). The dynein-
binding proteins LIS1 and NDEL1 colocalize with dynein to the NE in prophase, and their absence delays
the transition into prometaphase in neural stem cells, probably by affecting NEBD57. Notably,
microtubule-dependent tearing is not essential for the disruption of the NE, as NEBD is delayed rather
than blocked in nocodazole-treated cells17–19. Figure 3 | Nuclear envelope breakdown during ‘open’
mitosis. a | The images show HeLa cells in which the inner nuclear membrane (INM, green; stained by
green fluorescent protein fused to lamina-associated protein 2β), DNA (blue; stained with Hoechst) and
microtubules (red; stained by red fluorescent protein–α-tubulin) are visualized in G2, prophase and
metaphase. Scale bars, 10 μm. b | At the end of G2 phase, the activation of mitotic kinases, including the
master mitotic regulator cyclin-dependent kinase 1 (CDK1), triggers entry into prophase, which is
associated with a series of events that include the start of chromatin condensation, formation of
microtubule asters around centrosomes and centrosome separation. Microtubules that are attached to
the nuclear envelope (NE) in conjunction with the minus-end-directed motor dynein lead to NE
invaginations around centrosomes and to the formation of holes on the opposing site of the NE. At the
same time, nuclear pore complex (NPC) disassembly commences and is probably caused by the
phosphorylation of nucleoporins. The transition into prometaphase is marked by the loss of the NE
permeability barrier. Phosphorylation of nuclear lamins and INM proteins by CDK1, protein kinase C
(PKC) and probably other kinases results in lamina disassembly and allows for the retraction of NE
membranes into the endoplasmic reticulum (ER). In metaphase, most soluble components of the NE are
dispersed throughout the cytoplasm, whereas INM proteins reside in the tubular mitotic ER. ELYS is
known as MEL-28 in Caenorhabditis elegans. MTOC, microtubule-organizing centre; NEK2, NimA-related
kinase 2; PLK1, polo-like kinase 1; TOPOIIα, topoisomerase IIα. REVIEWS NATURE REVIEWS |
MOLECULAR CELL BIOLOGY VOLUME 10 | MARCH 2009 | 181 © 2009 Macmillan Publishers Limited. All
rights reserved Green - INM (lamin) Blue - DNA Red - microtubules Güttinger, Laurell & Kutay (2009)
Nature Reviews Molecular Cell Biology Breakdown of the Nuclear Envelope Güttinger, Laurell & Kutay
(2009) Nature Reviews Molecular Cell Biology Nature Reviews | Molecular Cell Biology Lamins NUP107–
160 and ELYS Transmembrane nucleoporins INM proteins MTOCs Chromatin Microtubules Kinetochore
Chromatin compaction KID, Aurora B? a b Early anaphase Late anaphase Telophase Completion of NPC
assembly Lamina assembly Formation of prepores and recruitment of membrane tubules NUP107–160
and ELYS, RanGTP NE reformation • INM proteins bind DNA • NE sheet formation removal of reticulons
• Membrane closure E3 ubiquitin ligase The last enzyme in a cascade of enzymes (E1, E2 and E3) that
mediates the attachment of mono- or polyubiquitin to target proteins. E3 enzymes are binding platforms
for E2 ligases and substrate proteins and thereby confer specificity to the ubiquitylation reaction.
Annulate lamellae Stacks of membrane cisternae, usually localized in the cytoplasm, that are densely
packed with nuclear pore complexes. during anaphase, although the exact timing and the phosphatases
that are responsible for this are unknown. A third contribution to the control of chromatin association of
the NUP107–160 complex has been revealed by the discovery of its chromatin-targeting factor ELYS
(MEL-28 in C. elegans), which is required to confine postmitotic NPC assembly to the surface of
chromatin80,132,133. Consistently, RNA interference-mediated knockdown of vertebrate ELYS results in
a reduction in the number of NPCs in the NE accompanied by an increase in ectopic NPC assembly in
annulate lamellae as a result of uncoupling NPC assembly from chromatin132,133. Likewise, mutations
in MEL-28 lead to defects in NE morphology and NPC assembly in C. elegans80,134. The binding of ELYS
to chromatin depends on an AT-hook DNA-binding motif in the carboxyl terminus of ELYS and occurs at
AT-rich chromatin regions135,136. But what controls ELYS association with chromatin? Again, several
mechanisms can be envisioned to collectively control this reaction. Spatial control is brought about by
the RanGTP-dependent recruitment of ELYS to chromatin133,134. Temporal regulation might depend on
the dephosphorylation of ELYS or chromatin proteins during anaphase as well as on the timely exposure
of AT-rich DNA regions during chromatin decondensation. Subsequent events in NPC assembly include
the association of membranes and other soluble nucleoporins with the chromatin-associated prepore,
to produce a Figure 5 | Nuclear envelope reassembly after mitosis. a | The images show HeLa cells in
which the inner nuclear membrane (INM, green; stained by green fluorescent protein fused to lamina-
associated protein 2β (LAP2β)), DNA (blue; stained with Hoechst) and microtubules (red; stained by red
fluorescent protein–α-tubulin) are visualized in early anaphase, late anaphase and telophase. Scale bars,
10 μm. b | In anaphase, separating chromatin masses are compacted by the action of the DNA-binding
chromokinesin KID (also known as KIF22 and kinesin 10). At this stage of mitosis, INM proteins, such as
LAP2β, are still dispersed in the tubular mitotic endoplasmic reticulum (ER). Nuclear pore complex (NPC)
assembly is initiated during anaphase by the recruitment of nucleoporin NUP107–160 complexes
through ELYS (MEL-28 in Caenorhabditis elegans) to chromatin, resulting in the formation of chromatin-
bound ‘prepores’. This requires the RanGTP-dependent liberation of NUP107–160 complexes from an
inhibitory association with importin-β. During late anaphase, ER membrane tubules start binding to the
chromatin surface. What mediates the initial attachment of the tubules to chromatin is unknown.During
telophase, the retraction of membrane-bending proteins (reticulons) into the peripheral ER allows the
remodelling of ER tubules into flattened membrane sheets on the chromatin surface. Binding of INM
proteins to DNA/chromatin supports the attachment of membrane sheets to chromatin. The first traces
of lamins can be detected on chromatin at this stage. NPC formation is completed by the step-wise
recruitment of further NPC constituents and the nuclear envelope (NE) is sealed. Finally, transport-
competent NPCs allow for the nuclear import of lamins to complete the assembly of the nuclear lamina.
MTOC, microtubule-organizing centre. REVIEWS 186 | MARCH 2009 | VOLUME 10
www.nature.com/reviews/molcellbio © 2009 Macmillan Publishers Limited. All rights reserved
Reformation of the Nuclear Envelope Green - INM (lamin) Blue - DNA Red - microtubules “To support
growth, a single nucleus must import approximately 560,000 ribosomal proteins and export
approximately 14,000 ribosomal subunits every minute.” CHAPTER 12 • Control of Gene Expression 462
The Nuclear Pore Complex and Its Role in Nucleocytoplasmic Trafficking The nuclear envelope is the
barrier between the nucleus and cytoplasm, and nuclear pores are the gateways across that barrier.
Unlike the plasma membrane, which prevents passage of macromolecules between the cytoplasm and
the extracellular space, the nuclear envelope is a hub of activity for the movement of RNAs and proteins
in both directions between the nucleus and cytoplasm. The replication and transcription of genetic
material within the nucleus require the participation of large numbers of proteins that are synthesized in
the cytoplasm and transported across the nuclear envelope. Conversely, mRNAs, tRNAs, and ribosomal
subunits that are manufactured in the nucleus must be transported through the nuclear envelope in the
opposite direction. Some components, such as the snRNAs of the spliceosome (page 426), move in both
directions; they are synthesized in the nucleus, assembled into RNP particles in the cytoplasm, and then
shipped back to the nucleus where they function in mRNA processing. To appreciate the magnitude of
the traffic between the two major cellular compartments, consider a HeLa cell, which is estimated to
contain about 10,000,000 ribosomes. To support its growth, a single HeLa cell nucleus must import
approximately 560,000 ribosomal proteins and export approximately 14,000 ribosomal subunits every
minute. How do all of these materials pass through the nuclear envelope? In one early approach, a
suspension of tiny gold particles was injected into cells and passage of the material through the nuclear
envelope was observed with the electron microscope. As illustrated in FIGURE 12.8 a , b , these particles
move from the cytoplasm into the nucleus by passing single‐file through the center of the nuclear pores.
Electron micrographs of cells fixed in the normal course of their activities have also shown that
particulate material can pass through a nuclear pore. An example is shown in Figure 12.8 c , in which
granular material presumed to consist of a ribosomal subunit is seen squeezing through one of these
pores. Given the fact that materials as large as gold particles and ribosomal subunits can penetrate
nuclear pores, one might assume that these pores are merely open channels, but just the opposite is
true. Nuclear pores contain a doughnut‐shaped structure called the nuclear pore complex( NPC ) that
straddles the nuclear envelope, projecting into both the cytoplasm and nucleoplasm. The NPC is a huge,
supramolecular complex—15 to 30 times the mass of a ribosome—that exhibits octagonal symmetry
due to the eightfold repetition of a number of structures (Figure 12.10 ). Despite their considerable size
and complexity, NPCs contain only about 30 different proteins, called nucleoporins , which are largely
conserved between yeast and vertebrates. Each nucleoporin is present in multiple copies—8, 16, or 32
in number—in keeping with the octagonal symmetry of the structure. The structure of the NPC is seen in
the electron micrographs of FIGURE 12.9 and the models of FIGURE 12.10 . At the heart of the NPC is a
central channel, which is surrounded by a ring of nucleoporins whose rearrangements can change the
diameter of the opening from about 20 to 40 nm. The NPC is not a static structure, as evidenced by the
finding that many of its component proteins are replaced with new copies over a time period of seconds
to minutes. Recent studies suggest that this dynamic exchange of nucleoporins may play a role in
activating the transcription of chromatin that is associated with the NPC. Among the nucleoporins is a
subset of proteins that possess, within their amino acid sequence, a large number of phenylalanine‐
glycine repeats (FG, by their single letter names). The FG repeats are clustered in a particular region of
each molecule called the FG domain. Because of their unusual amino acid composition, the FG domains
possess a disordered structure (page 55) that gives them an extended and flexible organization. The FG
repeat‐containing nucleoporins are thought to line the central channel of the NPC with their filamentous
FG domains extending into the heart of the central channel. The FG domains form a hydrophobic
meshwork or sieve that blocks the free diffusion of larger macromolecules (greater than about 40,000
Daltons) between the nucleus and cytoplasm. In 1982, Robert Laskey and his co ‐workers at the Medical
Research Council of England found that nucleoplasmin, one of the more abundant nuclear proteins of
amphibian oocytes, contains a stretch of amino acids near its C‐terminus that functions as a nuclear
localization signal( NLS ) . This sequence enables a protein to pass through the nuclear pores and enter
the nucleus. The best studied, or “classical,” NLSs consist of one or two short stretches of positively
charged amino acids. The T antigen encoded by the virus SV40, for FIGURE 12.8 The movement of
materials through the nuclear pore. ( a) Electron micrograph of the nuclear–cytoplasmic border of a frog
oocyte taken minutes after injection with gold particles that had been coated with a protein normally
found in the nucleus. These particles pass through the center of the nuclear pores (arrows) on their way
from the cytoplasm to the nucleus. ( b) At higher magnification, the gold particles are seen to be
clustered in a linear array within each pore. ( c) Electron micrograph of a section through the nuclear
envelope of an insect cell showing the movement of granular material (presumed to be a ribosomal
subunit) through a nuclear pore. SOURCE: (a, b) Courtesy of C. M. Feldherr; (c) From Barbara J. Stevens
and Hewson Swift, J. Cell Biol. 31:72, 1966, Fig. 23. Reproduced with permission of the Rockefeller
University Press. Cy (c) (a) (b) Karp-c12.indd 462 11/24/2015 12:30:50 AM The movement of materials
through the nuclear pore. (a) Electron micrograph of the nuclear–cytoplasmic border of a frog oocyte
taken minutes after injection with gold particles that had been coated with a protein normally found in
the nucleus. These particles pass through the center of the nuclear pores (arrows) on their way from the
cytoplasm to the nucleus. (b) At higher magnification, the gold particles are seen to be clustered in a
linear array within each pore. (c) Electron micrograph of a section through the nuclear envelope of an
insect cell showing the movement of granular material (presumed to be a ribosomal subunit) through a
nuclear pore. Karp et al. Ungricht & Kutay (2017) Nature Reviews Molecular Cell Biology Nuclear
Transport 12.2 • Structure of the Nuclear Envelope 463 (a) (b) (c) NEL FIGURE 12.9 Scanning electron
micrographs of the nuclear pore complex from isolated nuclear envelopes of an amphibian oocyte. ( a)
The cytoplasmic face of the nuclear envelope showing the peripheral cytoplasmic ring of the nuclear
pore complex. ( b) The nuclear face of the nuclear envelope showing the basket ‐like appearance of the
inner portion of the complex. ( c) The nuclear face of the envelope showing the distribution of the NPCs
and places where intact patches of the nuclear lamina (NEL) are retained. In all of these micrographs,
isolated nuclear envelopes were fixed, dehydrated, dried, and metal‐coated. SOURCE: (a–c) From M. W.
Goldberg and T. D. Allen, J. Cell Biol. 119:1431, 1992, Figures 1–3. Reproduced with permission of the
Rockefeller University Press. FIGURE 12.10 A model of a vertebrate nuclear pore complex (NPC). ( a)
Schematic representation of a vertebrate NPC as it is situated within the nuclear envelope. This
elaborate structure consists of several parts, including a scaffold and transmembrane ring that anchors
the complex to the nuclear envelope, a cytoplasmic and a nuclear ring, a nuclear basket, and eight
cytoplasmic filaments. The FG‐containing nucleoporins line a central channel with their disordered FG ‐
containing domains extending into the opening and forming a hydrophobic meshwork. (b) Three ‐
dimensional reconstruction of a portion of a nuclear pore complex showing the localization of individual
nucleoporin molecules within the structure. The FG nucleoporins are shown in green. Several
transmembrane nucleoporins span the pore membrane, forming an outer ring that anchors the NPC to
the nuclear envelope. SOURCE: (b) From Javier Fernandez‐Martinezand Michael P. Rout , Curr. Opin. Cell
Biol. 21 : 604 , 2009 , with permission from Elsevier. (b) FG nucleoporins Transmembrane nucleoporin
ring NE membrane protein ONM INM NE Cytoplasmic filaments Cytoplasm Nucleoplasm FG-repeat
domains of FG nucleoporins Central scaffold Transmembrane ring Cytoplasmic ring Outer nuclear
membrane Nuclear envelope Inner nuclear membrane Central channel Nuclear ring Nuclear basket (a)
example, contains an NLS identified as ‐Pro‐Lys‐Lys‐Lys‐Arg‐Lys‐ Val‐. If one of the basic amino acids in
this sequence is replaced by a nonpolar amino acid, the protein fails to localize to the nucleus.
Conversely, if this NLS is fused to a nonnuclear protein, such as serum albumin, and injected into the
cytoplasm, the modified protein becomes concentrated in the nucleus. Thus, targeting of proteins to the
nucleus is similar in principle to trafficking of other proteins that are destined for segregation within a
particular organelle, such as a mitochondrion or a peroxisome (Section 8.21). In all of these cases, the
proteins possess a specific “address” that is recognized by a specific receptor that mediates its transport
into the organelle. The study of nuclear transport has been a very active area of research, driven by the
development of in vitro systems capable of selectively importing proteins and RNPs into the nucleus.
Using these systems, researchers have identified a family of proteins that function as mobile transport
receptors , ferrying macromolecules across the nuclear envelope. Within this family, importins move
macromolecules from the cytoplasm into the nucleus and exportins move macromolecules in the
opposite direction. FIGURE 12.11 a depicts some of the major steps that occur during the nuclear import
of a protein, such as nucleoplasmin, that contains Karp-c12.indd 463 11/24/2015 12:30:51 AM 12.2 •
Structure of the Nuclear Envelope 463 (a) (b) (c) NEL FIGURE 12.9 Scanning electron micrographs of the
nuclear pore complex from isolated nuclear envelopes of an amphibian oocyte. ( a) The cytoplasmic face
of the nuclear envelope showing the peripheral cytoplasmic ring of the nuclear pore complex. ( b) The
nuclear face of the nuclear envelope showing the basket‐like appearance of the inner portion of the
complex. ( c) The nuclear face of the envelope showing the distribution of the NPCs and places where
intact patches of the nuclear lamina (NEL) are retained. In all of these micrographs, isolated nuclear
envelopes were fixed, dehydrated, dried, and metal‐coated. SOURCE: (a–c) From M. W. Goldberg and T.
D. Allen, J. Cell Biol. 119:1431, 1992, Figures 1–3. Reproduced with permission of the Rockefeller
University Press. FIGURE 12.10 A model of a vertebrate nuclear pore complex (NPC). ( a) Schematic
representation of a vertebrate NPC as it is situated within the nuclear envelope. This elaborate structure
consists of several parts, including a scaffold and transmembrane ring that anchors the complex to the
nuclear envelope, a cytoplasmic and a nuclear ring, a nuclear basket, and eight cytoplasmic filaments.
The FG‐containing nucleoporins line a central channel with their disordered FG ‐containing domains
extending into the opening and forming a hydrophobic meshwork. (b) Three ‐ dimensional
reconstruction of a portion of a nuclear pore complex showing the localization of individual nucleoporin
molecules within the structure. The FG nucleoporins are shown in green. Several transmembrane
nucleoporins span the pore membrane, forming an outer ring that anchors the NPC to the nuclear
envelope. SOURCE: (b) From Javier Fernandez‐Martinezand Michael P. Rout , Curr. Opin. Cell Biol. 21 :
604 , 2009 , with permission from Elsevier. (b) FG nucleoporins Transmembrane nucleoporin ring NE
membrane protein ONM INM NE Cytoplasmic filaments Cytoplasm Nucleoplasm FG-repeat domains of
FG nucleoporins Central scaffold Transmembrane ring Cytoplasmic ring Outer nuclear membrane
Nuclear envelope Inner nuclear membrane Central channel Nuclear ring Nuclear basket (a) example,
contains an NLS identified as ‐Pro‐Lys‐Lys‐Lys‐Arg‐Lys‐ Val‐. If one of the basic amino acids in this
sequence is replaced by a nonpolar amino acid, the protein fails to localize to the nucleus. Conversely, if
this NLS is fused to a nonnuclear protein, such as serum albumin, and injected into the cytoplasm, the
modified protein becomes concentrated in the nucleus. Thus, targeting of proteins to the nucleus is
similar in principle to trafficking of other proteins that are destined for segregation within a particular
organelle, such as a mitochondrion or a peroxisome (Section 8.21). In all of these cases, the proteins
possess a specific “address” that is recognized by a specific receptor that mediates its transport into the
organelle. The study of nuclear transport has been a very active area of research, driven by the
development of in vitro systems capable of selectively importing proteins and RNPs into the nucleus.
Using these systems, researchers have identified a family of proteins that function as mobile transport
receptors , ferrying macromolecules across the nuclear envelope. Within this family, importins move
macromolecules from the cytoplasm into the nucleus and exportins move macromolecules in the
opposite direction. FIGURE 12.11 a depicts some of the major steps that occur during the nuclear import
of a protein, such as nucleoplasmin, that contains Karp-c12.indd 463 11/24/2015 12:30:51 AM Scanning
electron micrographs of the nuclear pore complex from isolated nuclear envelopes of an amphibian
oocyte. (a) The cytoplasmic face of the nuclear envelope showing the peripheral cytoplasmic ring of the
nuclear pore complex. (b) The nuclear face of the nuclear envelope showing the basket-like appearance
of the inner portion of the complex. (c) The nuclear face of the envelope showing the distribution of the
NPCs and places where intact patches of the nuclear lamina (NEL) are retained. In all of these
micrographs, isolated nuclear envelopes were fixed, dehydrated, dried, and metal-coated. 12.2 •
Structure of the Nuclear Envelope 463 (a) (b) (c) NEL FIGURE 12.9 Scanning electron micrographs of the
nuclear pore complex from isolated nuclear envelopes of an amphibian oocyte. ( a) The cytoplasmic face
of the nuclear envelope showing the peripheral cytoplasmic ring of the nuclear pore complex. ( b) The
nuclear face of the nuclear envelope showing the basket‐like appearance of the inner portion of the
complex. ( c) The nuclear face of the envelope showing the distribution of the NPCs and places where
intact patches of the nuclear lamina (NEL) are retained. In all of these micrographs, isolated nuclear
envelopes were fixed, dehydrated, dried, and metal‐coated. SOURCE: (a–c) From M. W. Goldberg and T.
D. Allen, J. Cell Biol. 119:1431, 1992, Figures 1–3. Reproduced with permission of the Rockefeller
University Press. FIGURE 12.10 A model of a vertebrate nuclear pore complex (NPC). ( a) Schematic
representation of a vertebrate NPC as it is situated within the nuclear envelope. This elaborate structure
consists of several parts, including a scaffold and transmembrane ring that anchors the complex to the
nuclear envelope, a cytoplasmic and a nuclear ring, a nuclear basket, and eight cytoplasmic filaments.
The FG‐containing nucleoporins line a central channel with their disordered FG ‐containing domains
extending into the opening and forming a hydrophobic meshwork. (b) Three ‐ dimensional
reconstruction of a portion of a nuclear pore complex showing the localization of individual nucleoporin
molecules within the structure. The FG nucleoporins are shown in green. Several transmembrane
nucleoporins span the pore membrane, forming an outer ring that anchors the NPC to the nuclear
envelope. SOURCE: (b) From Javier Fernandez‐Martinezand Michael P. Rout , Curr. Opin. Cell Biol. 21 :
604 , 2009 , with permission from Elsevier. (b) FG nucleoporins Transmembrane nucleoporin ring NE
membrane protein ONM INM NE Cytoplasmic filaments Cytoplasm Nucleoplasm FG-repeat domains of
FG nucleoporins Central scaffold Transmembrane ring Cytoplasmic ring Outer nuclear membrane
Nuclear envelope Inner nuclear membrane Central channel Nuclear ring Nuclear basket (a) example,
contains an NLS identified as ‐Pro‐Lys‐Lys‐Lys‐Arg‐Lys‐ Val‐. If one of the basic amino acids in this
sequence is replaced by a nonpolar amino acid, the protein fails to localize to the nucleus. Conversely, if
this NLS is fused to a nonnuclear protein, such as serum albumin, and injected into the cytoplasm, the
modified protein becomes concentrated in the nucleus. Thus, targeting of proteins to the nucleus is
similar in principle to trafficking of other proteins that are destined for segregation within a particular
organelle, such as a mitochondrion or a peroxisome (Section 8.21). In all of these cases, the proteins
possess a specific “address” that is recognized by a specific receptor that mediates its transport into the
organelle. The study of nuclear transport has been a very active area of research, driven by the
development of in vitro systems capable of selectively importing proteins and RNPs into the nucleus.
Using these systems, researchers have identified a family of proteins that function as mobile transport
receptors , ferrying macromolecules across the nuclear envelope. Within this family, importins move
macromolecules from the cytoplasm into the nucleus and exportins move macromolecules in the
opposite direction. FIGURE 12.11 a depicts some of the major steps that occur during the nuclear import
of a protein, such as nucleoplasmin, that contains Karp-c12.indd 463 11/24/2015 12:30:51 AM Viewed
from the cytoplasm Viewed from the nucleus Viewed from the nucleus Karp et al (a) (b) (c) Nuclear pore
complex (NPC) Huge, supramolecular complex - 15 to 30 times the mass of a ribosome (2.7 MDa)
Exhibits octagonal symmetry Can rearrange to change diameter of the opening from about 20 to 40 nm.
Contain only about 30 different proteins (nucleoporins), which are largely conserved between yeast and
vertebrates. Nucleoporins lining the inner cavity contain stretches of Phe-Gly (FG) repeats Clustered in a
particular region of each nucleoporin to form the FG domain. Possess a disordered structure that gives
them an extended and flexible organization. The FG domains form a hydrophobic meshwork or sieve
that blocks the free diffusion of larger macromolecules (greater than about 40,000 Daltons) between the
nucleus and cytoplasm. Density of nuclear pore complexes correlates with nuclear activity - typical cells
contain 3000–4000 complexes 12.2 • Structure of the Nuclear Envelope 463 (a) (b) (c) NEL FIGURE 12.9
Scanning electron micrographs of the nuclear pore complex from isolated nuclear envelopes of an
amphibian oocyte. ( a) The cytoplasmic face of the nuclear envelope showing the peripheral cytoplasmic
ring of the nuclear pore complex. ( b) The nuclear face of the nuclear envelope showing the basket ‐like
appearance of the inner portion of the complex. ( c) The nuclear face of the envelope showing the
distribution of the NPCs and places where intact patches of the nuclear lamina (NEL) are retained. In all
of these micrographs, isolated nuclear envelopes were fixed, dehydrated, dried, and metal ‐coated.
SOURCE: (a–c) From M. W. Goldberg and T. D. Allen, J. Cell Biol. 119:1431, 1992, Figures 1–3.
Reproduced with permission of the Rockefeller University Press. FIGURE 12.10 A model of a vertebrate
nuclear pore complex (NPC). ( a) Schematic representation of a vertebrate NPC as it is situated within
the nuclear envelope. This elaborate structure consists of several parts, including a scaffold and
transmembrane ring that anchors the complex to the nuclear envelope, a cytoplasmic and a nuclear
ring, a nuclear basket, and eight cytoplasmic filaments. The FG‐containing nucleoporins line a central
channel with their disordered FG‐containing domains extending into the opening and forming a
hydrophobic meshwork. (b) Three‐ dimensional reconstruction of a portion of a nuclear pore complex
showing the localization of individual nucleoporin molecules within the structure. The FG nucleoporins
are shown in green. Several transmembrane nucleoporins span the pore membrane, forming an outer
ring that anchors the NPC to the nuclear envelope. SOURCE: (b) From Javier Fernandez ‐Martinezand
Michael P. Rout , Curr. Opin. Cell Biol. 21 : 604 , 2009 , with permission from Elsevier. (b) FG
nucleoporins Transmembrane nucleoporin ring NE membrane protein ONM INM NE Cytoplasmic
filaments Cytoplasm Nucleoplasm FG-repeat domains of FG nucleoporins Central scaffold
Transmembrane ring Cytoplasmic ring Outer nuclear membrane Nuclear envelope Inner nuclear
membrane Central channel Nuclear ring Nuclear basket (a) example, contains an NLS identified as ‐Pro ‐
Lys‐Lys‐Lys‐Arg‐Lys‐ Val‐. If one of the basic amino acids in this sequence is replaced by a nonpolar
amino acid, the protein fails to localize to the nucleus. Conversely, if this NLS is fused to a nonnuclear
protein, such as serum albumin, and injected into the cytoplasm, the modified protein becomes
concentrated in the nucleus. Thus, targeting of proteins to the nucleus is similar in principle to trafficking
of other proteins that are destined for segregation within a particular organelle, such as a
mitochondrion or a peroxisome (Section 8.21). In all of these cases, the proteins possess a specific
“address” that is recognized by a specific receptor that mediates its transport into the organelle. The
study of nuclear transport has been a very active area of research, driven by the development of in vitro
systems capable of selectively importing proteins and RNPs into the nucleus. Using these systems,
researchers have identified a family of proteins that function as mobile transport receptors , ferrying
macromolecules across the nuclear envelope. Within this family, importins move macromolecules from
the cytoplasm into the nucleus and exportins move macromolecules in the opposite direction. FIGURE
12.11 a depicts some of the major steps that occur during the nuclear import of a protein, such as
nucleoplasmin, that contains Karp-c12.indd 463 11/24/2015 12:30:51 AM Nuclear pore complex (NPC)
Active transport through the NPC Synthesis NUPs b c a HP1 ER Torsin LULL1 LAP1 LAD A-type lamins B-
type lamins BAF LEM Import Export Intermediate filament Actin Microtubule Plectin Nesprin 3 Nesprin
1/2 LINC SUN1/2 Emerin NPC ONM PNS INM Dynein LBR Importin RAN•GTP Model I: Membrane fusion
occurs before NPC assembly Diffusion Retention Model III: Maturation of the NPC core occurs before
membrane fusion Nature Reviews | Molecular Cell Biology Model II: Pre-NPC assembly is followed by
membrane fusion and NPC maturation NPC scaffold Y complex Inner ring complex Ribosome RNA
Nascent polypeptide Figure 1 |Nuclear envelope architecture and the integration of new components.
a|^|6Je nucleaT enXeloRe N'
consists of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM), which form a
specialized membrane sheet of the endoplasmic reticulum (ER) that is attached to chromatin (dark blue).
On the nuclear side, a network of nuclear lamins (pink) and integral membrane proteins provides
mechanical support to the NE and contributes to chromatin organization by ancJoTinI tJe socalled
laminaassociated domains .#Ds
6Je lamin $ TeceRtoT .$4
HoT eZamRle
 tetJeTs JeteTocJTomatin to the NE by binding to modified histones and heterochromatin protein 1
(HP1). LAP2, emerin, MAN1 (LEM)-domain proteins associate YitJ nucleaT lamins and tJe
cJTomatinassociated DaTTieTtoautointeITation HactoT $#(
6Je nucleus is connected to tJe c[tosMeleton D[ tJe linMeT oH nucleosMeleton and c[tosMeleton .+NC
comRleZes tJat sRan tJe N'5ad7NC57N
domaincontaininI RTotein tTimeTs oH 57NoT 57NeZRTessed in most cells
Dind to tJe tails oH nesRTins in the perinuclear space (PNS). In the cytoplasm, nesprins interact with
actin, intermediate filaments (via plectin) and micTotuDules Xia moleculaT motoTs
NucleaT RoTe comRleZes NPCs
alloY HoT tJe selectiXe
 TeceRtoTmediated imRoTt and|eZRoTt oH macTomolecules and tJe diHHusion oH metaDolites and ions
DetYeen tJe nucleus and tJe c[toRlasm 6Jese laTIe RToteinaceous cJannels aTe Duilt D[ multiRles oH
aRRToZimatel[ diHHeTent nucleoRoTins N7Ps
tJat aTe oTIani\ed into a macTomoleculaT assemDl[ oH eiIJtHold Totational s[mmetT[6Je NPC scaHHold
see inset'lectTon /icToscoR[ Data $anM '/D$
'/D (REF. 210)
is HoTmed oH eiIJt sRoMes
eacJ consistinI oH HouT ; comRleZes N7PsN7PsuDcomRleZ
tYo RTotomeTs colouTed in [elloY
and HouT inneT TinI suDcomRleZes N7PsN7P suDcomRleZ
ITeen
####6Pases oH the torsin family and their cofactors lamina-associated protein 1 (LAP1) or lumenal
domain-like LAP1 (LULL1) form protein comRleZes in tJe PN5 and '4 lumen
TesRectiXel[
 and ma[ suRRoTt N' TemodellinI eXentsb|^|NeYl[ s[ntJesi\ed +N/destined memDTane RToteins aTe
inseTted into tJe '4s1N/ netYoTM
 in YJicJ tJe[ distTiDute D[ diHHusion PassaIe tJTouIJ tJe NPC to tJe +N/ is onl[ RossiDle HoT
tTansmemDTane RToteins tJat JaXe eZtTalumenal domains smalleT tJan aRRToZimatel[ MDa6Je
accumulation oH RToteins at tJe +N/ is dTiXen D[ tJeiT Tetention on cJTomatin oT tJe lamina meshwork.
INM-associated peripheral proteins and soluble nucleoporins reach the nuclear interior by receptor-
mediated import that is dependent on importins and nuclear RAN·)6Pc|^|#ssemDl[ oH neY NPCs in tJe
N' ma[ eitJeT occuT at a|staDili\ed memDTane RoTe tJat is HoTmed aHteT +N/s1N/ Husion model|+
oT it can De initiated D[ tJe HoTmation oH immatuTe|RTeNPCs DeneatJ tJe +N/ model|++
5uDseSuentl[
tJese RTeNPCs Yould De emDedded into tJe N' and tTiIIeT|+N/s1N/|Husion to comRlete matuTation
HTom tJe c[toRlasmic side#lteTnatiXel[
RTeNPCs miIJt matuTe HuTtJeT DeHoTe +N/s1N/ Husion is elicited model|+++
REVIEWS 230 | APRIL 2017 | VOLUME 18 www.nature.com/nrm ǟ ƐƎƏƗ !,(++- 4 +(2'#12
(,(3#"Ʀ /13 .$ /1(-%#1 341#ƥ ++ 1(%'32 1#2#15#"ƥ ǟ ƐƎƏƗ !,(++- 4 +(2'#12
(,(3#"Ʀ /13 .$ /1(-%#1 341#ƥ ++ 1(%'32 1#2#15#"ƥ • Selectivity and transport relies on nuclear
localization and export signals • NLSs is a sequence or patch • NLS has sequences that are rich in the
positively charged lysines/arginines • NES has sequences rich in hydrophobic residues Nuclear
Localization Signals and Nuclear Export Signals • Nuclear import receptors bind NLS and nucleoporins •
Soluble cytosolic proteins • FG-repeats on nucleoporins bind import receptors on the cytosolic side •
Nuclear export relies on nuclear export signals that bind nuclear export receptors • Nuclear export
receptors are structurally related to nuclear import receptors (karyopherins). Nuclear Import and Export
Receptors The core biochemistry of Ran is similar to that of many Ras-related small GTPases Ran’s
intrinsic rates of nucleotide exchange and hydrolysis are slow These reactions require a nucleotide
exchange factor GEF and a GTPase activating protein (GAP) Ran-GAP is cytosolic and binds to Ran-GTP
with high affinity and increase GTP hydrolysis Ran-GEF is a chromatin-associated nuclear protein This
asymmetric distribution of nucleotide exchange and hydrolysis enzymes across the nuclear envelope
predicts that Ran-GTP should be largely nuclear and Ran-GDP should be largely cytosolic. This
distribution plays a key role in determining the directionality of nuclear transport. Chook and Blobel
(1999) Nature Dasso (2001) Cell Nuclear Localisation Relies on a Gradient of the Small GTPase Ran
Ran:GTP Nuclear Localisation Relies on a Gradient of the Small GTPase Ran Nuclear envelope Ran GAP in
cytoplasm Ran GEF on chromosomes GTP Nuclear Import Cycle • Complex of NLS-bearing protein binds
nuclear transport receptor (NTR) in the cytoplasm • NTR binds FG repeats in the NPC • After
translocation through the NPC, RanGTP displaces the protein from the NTR • This causes release of
protein • NTR is bound to Ran GTP • NTR–Ran-GTP complex returns to the cytoplasm through the NPC •
RanGAP1 in the cytosol stimulates GTP hydrolysis on Ran-GTP • Releasing NTR for another import cycle
Journal of Cell Science (2016) Nuclear Export Cycle Journal of Cell Science (2016) • Nuclear export cycles
require the formation of a trimeric protein–NTR– Ran-GTP complex in the nucleus. • After NPC passage,
this complex dissociates due to Ran-GTP hydrolysis, releasing the cargo into the cytoplasm The Golgi
Apparatus 715 The Golgi Apparatus Consists of an Ordered Series of Compartments Because it could be
selectively visualized by silver stains, the Golgi apparatus was one of the frst organelles described by
early light microscopists. It consists of a collection of fattened, membrane-enclosed compartments
called cisternae, that somewhat resemble a stack of pita breads. Each Golgi stack typically consists of
four to six cisternae (Figure 13–26), although some unicellular fagellates can have more than 20. In
animal cells, tubular connections between corresponding cisternae link many stacks, thus forming a
single complex, which is usually located near the cell nucleus and close to the centrosome (Figure 13–
27A). Tis localization depends on microtubules. If microtubules are experimentally depolymerized, the
Golgi apparatus reorganizes into individual stacks that are found throughout the cytoplasm, adjacent to
ER exit sites. Some cells, including most plant cells, have hundreds of individual Golgi stacks dispersed
throughout the cytoplasm where they are typically found adjacent to ER exit sites (Figure 13–27B).
TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS cis Golgi network (CGN) cis cisterna medial
cisterna trans cisterna trans Golgi network (TGN) secretory vesicle (A) cis FACE Golgi vesicle trans FACE
nuclear envelope rough ER vesicular tubular clusters (B) 1 µm MBoC6 m13.25/13.25 Figure 13–26 The
Golgi apparatus. (A) Three-dimensional reconstruction from electron micrographs of the Golgi apparatus
in a secretory animal cell. The cis face of the Golgi stack is that closest to the ER. (B) A thin-section
electron micrograph of an animal cell. In plant cells, the Golgi apparatus is generally more distinct and
more clearly separated from other intracellular membranes than in animal cells. (A, redrawn from A.
Rambourg and Y. Clermont, Eur. J. Cell Biol. 51:189–200, 1990. With permission from Wissenschaftliche
Verlagsgesellschaft; B, courtesy of Brij J. Gupta.) (A) (B) MBoC6 m13.26/13.26 Figure 13–27 Localization
of the Golgi apparatus in animal and plant cells. (A) The Golgi apparatus in a cultured fibroblast stained
with a fluorescent antibody that recognizes a Golgi resident protein (bright orange). The Golgi apparatus
is polarized, facing the direction in which the cell was crawling before fixation. (B) The Golgi apparatus in
a plant cell that is expressing a fusion protein consisting of a resident Golgi enzyme fused to green
fluorescent protein. (A, courtesy of John Henley and Mark McNiven; B, courtesy of Chris Hawes.) A.
Three-dimensional reconstruction from electron micrographs of the golgi apparatus in a secretory
animal cell. The cis face of the golgi stack is that closest to the er. B. A thin-section electron micrograph
of an animal cell. In plant cells, the golgi apparatus is generally more distinct and more clearly separated
from other intracellular membranes than in animal cells. Transport of proteins tells us of the secretory
pathway Boncompain et al. (2012) Nature Methods 716 Chapter 13: Intracellular Membrane Traffic
During their passage through the Golgi apparatus, transported molecules undergo an ordered series of
covalent modifcations. Each Golgi stack has two distinct faces: a cis face (or entry face) and a trans face
(or exit face). Both cis and trans faces are closely associated with special compartments, each composed
of a network of interconnected tubular and cisternal structures: the cis Golgi network (CGN) and the
trans Golgi network (TGN), respectively. Te CGN is a collection of fused vesicular tubular clusters arriving
from the ER. Proteins and lipids enter the cis Golgi network and exit from the trans Golgi network,
bound for the cell surface or another compartment. Both networks are important for protein sorting:
proteins entering the CGN can either move onward in the Golgi apparatus or be returned to the ER.
Similarly, proteins exiting from the TGN move onward and are sorted according to their next
destination: endosomes, secretory vesicles, or the cell surface. Tey also can be returned to an earlier
compartment. Some membrane proteins are retained in the part of the Golgi apparatus where they
function. As described in Chapter 12, a single species of N-linked oligosaccharide is attached en bloc to
many proteins in the ER and then trimmed while the protein is still in the ER. Te oligosaccharide
intermediates created by the trimming reactions serve to help proteins fold and to help transport
misfolded proteins to the cytosol for degradation in proteasomes. Tus, they play an important role in
controlling the quality of proteins exiting from the ER. Once these ER functions have been fulflled, the
cell reutilizes the oligosaccharides for new functions. Tis begins in the Golgi apparatus, which generates
the heterogeneous oligosaccharide structures seen in mature proteins. After arrival in the CGN, proteins
enter the frst of the Golgi processing compartments (the cis Golgi cisternae). Tey then move to the next
compartment (the medial cisternae) and fnally to the trans cisternae, where glycosylation is completed.
Te lumen of the trans cisternae is thought to be continuous with the TGN, the place where proteins are
segregated into diferent transport packages and dispatched to their fnal destinations. Te oligosaccharide
processing steps occur in an organized sequence in the Golgi stack, with each cisterna containing a
characteristic mixture of processing enzymes. Proteins are modifed in successive stages as they move
from cisterna to cisterna across the stack, so that the stack forms a multistage processing unit.
Investigators discovered the functional diferences between the cis, medial, and trans subdivisions of the
Golgi apparatus by localizing the enzymes involved in processing N-linked oligosaccharides in distinct
regions of the organelle, both by physical fractionation of the organelle and by labeling the enzymes in
electron microscope sections with antibodies (Figure 13–28). Te removal of mannose and the addition of
N-acetylglucosamine, for example, occur in the cis and medial cisternae, while the addition of galactose
and sialic acid occurs in the trans cisterna and trans Golgi network. Figure 13–29 summarizes the
functional compartmentalization of the Golgi apparatus. Oligosaccharide Chains Are Processed in the
Golgi Apparatus Whereas the ER lumen is full of soluble lumenal resident proteins and enzymes, the
resident proteins in the Golgi apparatus are all membrane bound, as the enzymatic reactions apparently
occur entirely on membrane surfaces. All of the Golgi glycosidases and glycosyl transferases, for
example, are single-pass transmembrane proteins, many of which are organized in multienzyme
complexes. (A) (B) (C) (D) 1 µm MBoC6 m13.27/13.27 Figure 13–28 Molecular compartmentalization of
the Golgi apparatus. A series of electron micrographs shows the Golgi apparatus (A) unstained, (B)
stained with osmium, which preferentially labels the cisternae of the cis compartment, and (C and D)
stained to reveal the location of specific enzymes. Nucleoside diphosphatase is found in the trans Golgi
cisternae (C), while acid phosphatase is found in the trans Golgi network (D). Note that usually more
than one cisterna is stained. The enzymes are therefore thought to be highly enriched rather than
precisely localized to a specific cisterna. (Courtesy of Daniel S. Friend.) Molecular compartmentalization
of the Golgi apparatus Unstained Nucleoside diphosphatase 716 Chapter 13: Intracellular Membrane
Traffic During their passage through the Golgi apparatus, transported molecules undergo an ordered
series of covalent modifcations. Each Golgi stack has two distinct faces: a cis face (or entry face) and a
trans face (or exit face). Both cis and trans faces are closely associated with special compartments, each
composed of a network of interconnected tubular and cisternal structures: the cis Golgi network (CGN)
and the trans Golgi network (TGN), respectively. Te CGN is a collection of fused vesicular tubular clusters
arriving from the ER. Proteins and lipids enter the cis Golgi network and exit from the trans Golgi
network, bound for the cell surface or another compartment. Both networks are important for protein
sorting: proteins entering the CGN can either move onward in the Golgi apparatus or be returned to the
ER. Similarly, proteins exiting from the TGN move onward and are sorted according to their next
destination: endosomes, secretory vesicles, or the cell surface. Tey also can be returned to an earlier
compartment. Some membrane proteins are retained in the part of the Golgi apparatus where they
function. As described in Chapter 12, a single species of N-linked oligosaccharide is attached en bloc to
many proteins in the ER and then trimmed while the protein is still in the ER. Te oligosaccharide
intermediates created by the trimming reactions serve to help proteins fold and to help transport
misfolded proteins to the cytosol for degradation in proteasomes. Tus, they play an important role in
controlling the quality of proteins exiting from the ER. Once these ER functions have been fulflled, the
cell reutilizes the oligosaccharides for new functions. Tis begins in the Golgi apparatus, which generates
the heterogeneous oligosaccharide structures seen in mature proteins. After arrival in the CGN, proteins
enter the frst of the Golgi processing compartments (the cis Golgi cisternae). Tey then move to the next
compartment (the medial cisternae) and fnally to the trans cisternae, where glycosylation is completed.
Te lumen of the trans cisternae is thought to be continuous with the TGN, the place where proteins are
segregated into diferent transport packages and dispatched to their fnal destinations. Te oligosaccharide
processing steps occur in an organized sequence in the Golgi stack, with each cisterna containing a
characteristic mixture of processing enzymes. Proteins are modifed in successive stages as they move
from cisterna to cisterna across the stack, so that the stack forms a multistage processing unit.
Investigators discovered the functional diferences between the cis, medial, and trans subdivisions of the
Golgi apparatus by localizing the enzymes involved in processing N-linked oligosaccharides in distinct
regions of the organelle, both by physical fractionation of the organelle and by labeling the enzymes in
electron microscope sections with antibodies (Figure 13–28). Te removal of mannose and the addition of
N-acetylglucosamine, for example, occur in the cis and medial cisternae, while the addition of galactose
and sialic acid occurs in the trans cisterna and trans Golgi network. Figure 13–29 summarizes the
functional compartmentalization of the Golgi apparatus. Oligosaccharide Chains Are Processed in the
Golgi Apparatus Whereas the ER lumen is full of soluble lumenal resident proteins and enzymes, the
resident proteins in the Golgi apparatus are all membrane bound, as the enzymatic reactions apparently
occur entirely on membrane surfaces. All of the Golgi glycosidases and glycosyl transferases, for
example, are single-pass transmembrane proteins, many of which are organized in multienzyme
complexes. (A) (B) (C) (D) 1 µm MBoC6 m13.27/13.27 Figure 13–28 Molecular compartmentalization of
the Golgi apparatus. A series of electron micrographs shows the Golgi apparatus (A) unstained, (B)
stained with osmium, which preferentially labels the cisternae of the cis compartment, and (C and D)
stained to reveal the location of specific enzymes. Nucleoside diphosphatase is found in the trans Golgi
cisternae (C), while acid phosphatase is found in the trans Golgi network (D). Note that usually more
than one cisterna is stained. The enzymes are therefore thought to be highly enriched rather than
precisely localized to a specific cisterna. (Courtesy of Daniel S. Friend.) 716 Chapter 13: Intracellular
Membrane Traffic During their passage through the Golgi apparatus, transported molecules undergo an
ordered series of covalent modifcations. Each Golgi stack has two distinct faces: a cis face (or entry face)
and a trans face (or exit face). Both cis and trans faces are closely associated with special compartments,
each composed of a network of interconnected tubular and cisternal structures: the cis Golgi network
(CGN) and the trans Golgi network (TGN), respectively. Te CGN is a collection of fused vesicular tubular
clusters arriving from the ER. Proteins and lipids enter the cis Golgi network and exit from the trans
Golgi network, bound for the cell surface or another compartment. Both networks are important for
protein sorting: proteins entering the CGN can either move onward in the Golgi apparatus or be
returned to the ER. Similarly, proteins exiting from the TGN move onward and are sorted according to
their next destination: endosomes, secretory vesicles, or the cell surface. Tey also can be returned to an
earlier compartment. Some membrane proteins are retained in the part of the Golgi apparatus where
they function. As described in Chapter 12, a single species of N-linked oligosaccharide is attached en bloc
to many proteins in the ER and then trimmed while the protein is still in the ER. Te oligosaccharide
intermediates created by the trimming reactions serve to help proteins fold and to help transport
misfolded proteins to the cytosol for degradation in proteasomes. Tus, they play an important role in
controlling the quality of proteins exiting from the ER. Once these ER functions have been fulflled, the
cell reutilizes the oligosaccharides for new functions. Tis begins in the Golgi apparatus, which generates
the heterogeneous oligosaccharide structures seen in mature proteins. After arrival in the CGN, proteins
enter the frst of the Golgi processing compartments (the cis Golgi cisternae). Tey then move to the next
compartment (the medial cisternae) and fnally to the trans cisternae, where glycosylation is completed.
Te lumen of the trans cisternae is thought to be continuous with the TGN, the place where proteins are
segregated into diferent transport packages and dispatched to their fnal destinations. Te oligosaccharide
processing steps occur in an organized sequence in the Golgi stack, with each cisterna containing a
characteristic mixture of processing enzymes. Proteins are modifed in successive stages as they move
from cisterna to cisterna across the stack, so that the stack forms a multistage processing unit.
Investigators discovered the functional diferences between the cis, medial, and trans subdivisions of the
Golgi apparatus by localizing the enzymes involved in processing N-linked oligosaccharides in distinct
regions of the organelle, both by physical fractionation of the organelle and by labeling the enzymes in
electron microscope sections with antibodies (Figure 13–28). Te removal of mannose and the addition of
N-acetylglucosamine, for example, occur in the cis and medial cisternae, while the addition of galactose
and sialic acid occurs in the trans cisterna and trans Golgi network. Figure 13–29 summarizes the
functional compartmentalization of the Golgi apparatus. Oligosaccharide Chains Are Processed in the
Golgi Apparatus Whereas the ER lumen is full of soluble lumenal resident proteins and enzymes, the
resident proteins in the Golgi apparatus are all membrane bound, as the enzymatic reactions apparently
occur entirely on membrane surfaces. All of the Golgi glycosidases and glycosyl transferases, for
example, are single-pass transmembrane proteins, many of which are organized in multienzyme
complexes. (A) (B) (C) (D) 1 µm MBoC6 m13.27/13.27 Figure 13–28 Molecular compartmentalization of
the Golgi apparatus. A series of electron micrographs shows the Golgi apparatus (A) unstained, (B)
stained with osmium, which preferentially labels the cisternae of the cis compartment, and (C and D)
stained to reveal the location of specific enzymes. Nucleoside diphosphatase is found in the trans Golgi
cisternae (C), while acid phosphatase is found in the trans Golgi network (D). Note that usually more
than one cisterna is stained. The enzymes are therefore thought to be highly enriched rather than
precisely localized to a specific cisterna. (Courtesy of Daniel S. Friend.) Osmium Acid phosphatase 716
Chapter 13: Intracellular Membrane Traffic During their passage through the Golgi apparatus,
transported molecules undergo an ordered series of covalent modifcations. Each Golgi stack has two
distinct faces: a cis face (or entry face) and a trans face (or exit face). Both cis and trans faces are closely
associated with special compartments, each composed of a network of interconnected tubular and
cisternal structures: the cis Golgi network (CGN) and the trans Golgi network (TGN), respectively. Te CGN
is a collection of fused vesicular tubular clusters arriving from the ER. Proteins and lipids enter the cis
Golgi network and exit from the trans Golgi network, bound for the cell surface or another
compartment. Both networks are important for protein sorting: proteins entering the CGN can either
move onward in the Golgi apparatus or be returned to the ER. Similarly, proteins exiting from the TGN
move onward and are sorted according to their next destination: endosomes, secretory vesicles, or the
cell surface. Tey also can be returned to an earlier compartment. Some membrane proteins are retained
in the part of the Golgi apparatus where they function. As described in Chapter 12, a single species of N-
linked oligosaccharide is attached en bloc to many proteins in the ER and then trimmed while the
protein is still in the ER. Te oligosaccharide intermediates created by the trimming reactions serve to
help proteins fold and to help transport misfolded proteins to the cytosol for degradation in
proteasomes. Tus, they play an important role in controlling the quality of proteins exiting from the ER.
Once these ER functions have been fulflled, the cell reutilizes the oligosaccharides for new functions. Tis
begins in the Golgi apparatus, which generates the heterogeneous oligosaccharide structures seen in
mature proteins. After arrival in the CGN, proteins enter the frst of the Golgi processing compartments
(the cis Golgi cisternae). Tey then move to the next compartment (the medial cisternae) and fnally to the
trans cisternae, where glycosylation is completed. Te lumen of the trans cisternae is thought to be
continuous with the TGN, the place where proteins are segregated into diferent transport packages and
dispatched to their fnal destinations. Te oligosaccharide processing steps occur in an organized
sequence in the Golgi stack, with each cisterna containing a characteristic mixture of processing
enzymes. Proteins are modifed in successive stages as they move from cisterna to cisterna across the
stack, so that the stack forms a multistage processing unit. Investigators discovered the functional
diferences between the cis, medial, and trans subdivisions of the Golgi apparatus by localizing the
enzymes involved in processing N-linked oligosaccharides in distinct regions of the organelle, both by
physical fractionation of the organelle and by labeling the enzymes in electron microscope sections with
antibodies (Figure 13–28). Te removal of mannose and the addition of N-acetylglucosamine, for
example, occur in the cis and medial cisternae, while the addition of galactose and sialic acid occurs in
the trans cisterna and trans Golgi network. Figure 13–29 summarizes the functional
compartmentalization of the Golgi apparatus. Oligosaccharide Chains Are Processed in the Golgi
Apparatus Whereas the ER lumen is full of soluble lumenal resident proteins and enzymes, the resident
proteins in the Golgi apparatus are all membrane bound, as the enzymatic reactions apparently occur
entirely on membrane surfaces. All of the Golgi glycosidases and glycosyl transferases, for example, are
single-pass transmembrane proteins, many of which are organized in multienzyme complexes. (A) (B) (C)
(D) 1 µm MBoC6 m13.27/13.27 Figure 13–28 Molecular compartmentalization of the Golgi apparatus. A
series of electron micrographs shows the Golgi apparatus (A) unstained, (B) stained with osmium, which
preferentially labels the cisternae of the cis compartment, and (C and D) stained to reveal the location of
specific enzymes. Nucleoside diphosphatase is found in the trans Golgi cisternae (C), while acid
phosphatase is found in the trans Golgi network (D). Note that usually more than one cisterna is stained.
The enzymes are therefore thought to be highly enriched rather than precisely localized to a specific
cisterna. (Courtesy of Daniel S. Friend.) Birth and Maintenance of the Golgi Apparatus 713 the electron
microscope (Figure 13–24A). Tese clusters constitute a compartment that is separate from the ER and
lacks many of the proteins that function in the ER. Tey are generated continually and function as
transport containers that bring material from the ER to the Golgi apparatus. Te clusters move quickly
along microtubules to the Golgi apparatus with which they fuse (Figure 13–24B and Movie 13.2). As
soon as vesicular tubular clusters form, they begin to bud of transport vesicles of their own. Unlike the
COPII-coated vesicles that bud from the ER, these vesicles are COPI-coated (see Figure 13–24A). COPI-
coated vesicles are unique in that the components that make up the inner and outer coat layers are
recruited as a preassembled complex, called coatomer. Tey function as a retrieval pathway, carrying
back ER resident proteins that have escaped, as well as proteins such as cargo receptors and SNAREs
that participated in the ER budding and vesicle fusion reactions. Tis retrieval process demonstrates the
exquisite control mechanisms that regulate coat assembly reactions. Te COPI coat assembly begins only
seconds after the COPII coats have been shed, and remains a mystery how this switch in coat assembly is
controlled. Te retrieval (or retrograde) transport continues as the vesicular tubular clusters move toward
the Golgi apparatus. Tus, the clusters continuously mature, gradually changing their composition as
selected proteins are returned to the ER. Te retrieval continues from the Golgi apparatus, after the
vesicular tubular clusters have delivered their cargo. The Retrieval Pathway to the ER Uses Sorting
Signals Te retrieval pathway for returning escaped proteins back to the ER depends on ER retrieval
signals. Resident ER membrane proteins, for example, contain signals that bind directly to COPI coats
and are thus packaged into COPI-coated transport vesicles for retrograde delivery to the ER. Te best-
characterized retrieval signal of this type consists of two lysines, followed by any two other amino acids,
at the extreme C-terminal end of the ER membrane protein. It is called a KKXX sequence, based on the
single-letter amino acid code. Soluble ER resident proteins, such as BiP, also contain a short ER retrieval
signal at their C-terminal end, but it is diferent: it consists of a Lys-Asp-Glu-Leu or a similar sequence. If
this signal (called the KDEL sequence) is removed from BiP by genetic engineering, the protein is slowly
secreted from the cell. If the signal is transferred to a protein that is normally secreted, the protein is
now efciently returned to the ER, where it accumulates. TRANSPORT FROM THE ER THROUGH THE
GOLGI APPARATUS vesicular tubular cluster ER (A) 0.2 µm microtubule cis Golgi network COPI coat
vesicular tubular cluster COPII coat ER motor protein retrieval transport (B) MBoC6 m13.23/13.23 Figure
13–24 Vesicular tubular clusters. (A) An electron micrograph of vesicular tubular clusters forming around
an exit site. Many of the vesicle-like structures seen in the micrograph are cross sections of tubules that
extend above and below the plane of this thin section and are interconnected. (B) Vesicular tubular
clusters move along microtubules to carry proteins from the ER to the Golgi apparatus. COPI-coated
vesicles mediate the budding of vesicles that return to the ER from these clusters (and from the Golgi
apparatus). (A, courtesy of William Balch.) 713 the electron microscope (Figure 13–24A). Tese clusters
constitute a compartment that is separate from the ER and lacks many of the proteins that function in
the ER. Tey are generated continually and function as transport containers that bring material from the
ER to the Golgi apparatus. Te clusters move quickly along microtubules to the Golgi apparatus with
which they fuse (Figure 13–24B and Movie 13.2). As soon as vesicular tubular clusters form, they begin
to bud of transport vesicles of their own. Unlike the COPII-coated vesicles that bud from the ER, these
vesicles are COPI-coated (see Figure 13–24A). COPI-coated vesicles are unique in that the components
that make up the inner and outer coat layers are recruited as a preassembled complex, called coatomer.
Tey function as a retrieval pathway, carrying back ER resident proteins that have escaped, as well as
proteins such as cargo receptors and SNAREs that participated in the ER budding and vesicle fusion
reactions. Tis retrieval process demonstrates the exquisite control mechanisms that regulate coat
assembly reactions. Te COPI coat assembly begins only seconds after the COPII coats have been shed,
and remains a mystery how this switch in coat assembly is controlled. Te retrieval (or retrograde)
transport continues as the vesicular tubular clusters move toward the Golgi apparatus. Tus, the clusters
continuously mature, gradually changing their composition as selected proteins are returned to the ER.
Te retrieval continues from the Golgi apparatus, after the vesicular tubular clusters have delivered their
cargo. The Retrieval Pathway to the ER Uses Sorting Signals Te retrieval pathway for returning escaped
proteins back to the ER depends on ER retrieval signals. Resident ER membrane proteins, for example,
contain signals that bind directly to COPI coats and are thus packaged into COPI-coated transport
vesicles for retrograde delivery to the ER. Te best-characterized retrieval signal of this type consists of
two lysines, followed by any two other amino acids, at the extreme C-terminal end of the ER membrane
protein. It is called a KKXX sequence, based on the single-letter amino acid code. Soluble ER resident
proteins, such as BiP, also contain a short ER retrieval signal at their C-terminal end, but it is diferent: it
consists of a Lys-Asp-Glu-Leu or a similar sequence. If this signal (called the KDEL sequence) is removed
from BiP by genetic engineering, the protein is slowly secreted from the cell. If the signal is transferred
to a protein that is normally secreted, the protein is now efciently returned to the ER, where it
accumulates. TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS vesicular tubular cluster ER
(A) 0.2 µm microtubule cis Golgi network COPI coat vesicular tubular cluster COPII coat ER motor protein
retrieval transport (B) MBoC6 m13.23/13.23 Figure 13–24 Vesicular tubular clusters. (A) An electron
micrograph of vesicular tubular clusters forming around an exit site. Many of the vesicle-like structures
seen in the micrograph are cross sections of tubules that extend above and below the plane of this thin
section and are interconnected. (B) Vesicular tubular clusters move along microtubules to carry proteins
from the ER to the Golgi apparatus. COPI-coated vesicles mediate the budding of vesicles that return to
the ER from these clusters (and from the Golgi apparatus). (A, courtesy of William Balch.) (A) An electron
micrograph of vesicular tubular clusters forming around the ER exit sites. (B) vesicular tubular clusters
move along microtubules to carry proteins from the ER to the Golgi apparatus. COPI-coated vesicles
mediate the budding of vesicles that return to the ER from these clusters and from the Golgi apparatus.
721 retrieves escaped ER and Golgi proteins and returns them to upstream compartments. Directional
fow could be achieved because forward-moving cargo molecules are selectively packaged into forward-
moving vesicles. Although both forward- and backward-moving vesicles would likely be COPI-coated, the
coats may contain diferent adaptor proteins that confer selectivity on the packaging of cargo molecules.
Alternatively, transport vesicles shuttling between Golgi cisternae might not be directional at all,
transporting cargo randomly back and forth; directional fow would then occur because of the continual
input to the cis cisterna and output from the trans cisterna. Te vesicle transport model is supported by
experiments that show that cargo molecules are present in small COPI-coated vesicles and that these
vesicles can deliver them to Golgi cisternae over large distances. In addition, when experimentally
aggregated membrane proteins are introduced into Golgi cisternae, they can be observed staying in
place, while soluble cargo, even if present as large aggregates, traverses the Golgi at normal rates. It is
likely that aspects of both models are true. A stable core of long-lasting cisternae might exist in the
center of each Golgi cisterna, while regions at the rim may undergo continuous maturation, perhaps
utilizing Rab cascades that change their identity. As matured pieces of the cisternae are formed, they
might break of and fuse with downstream cisternae by homotypic fusion mechanisms, taking large cargo
molecules with them. In addition, small COPI-coated vesicles might transport small cargo in the forward
direction and retrieve escaped Golgi enzymes and return them to their appropriate upstream cisternae.
Golgi Matrix Proteins Help Organize the Stack Te unique architecture of the Golgi apparatus depends on
both the microtubule cytoskeleton, as already mentioned, and cytoplasmic Golgi matrix proteins, which
form a scafold between adjacent cisternae and give the Golgi stack its structural integrity. Some of the
matrix proteins, called golgins, form long tethers composed of stif coiled-coil domains with interspersed
hinge regions. Golgins form a forest of tentacles that can extend 100–400 nm from the surface of the
Golgi stack. Tey are thought to help retain Golgi transport vesicles close to the organelle through
interactions with Rab proteins (Figure 13–36). When the cell prepares to divide, mitotic protein kinases
phosphorylate the Golgi matrix proteins, causing the Golgi TRANSPORT FROM THE ER THROUGH THE
GOLGI APPARATUS (A) CISTERNAL MATURATION MODEL (B) VESICLE TRANSPORT MODEL ER CGN TGN
cisternae cis trans medial vesicular tubular cluster MBoC6 m13.35/13.35 matrix proteins Figure 13–35
Two possible models explaining the organization of the Golgi apparatus and how proteins move through
it. It is likely that the transport through the Golgi apparatus in the forward direction (red arrows)
involves elements of both models. (A) According to the cisternal maturation model, each Golgi cisterna
matures as it migrates outward through the stack. At each stage, the Golgi resident proteins that are
carried forward in a maturing cisterna are moved backward to an earlier compartment in COPI-coated
vesicles. When a newly formed cisterna moves to a medial position, for example, “leftover” cis Golgi
enzymes would be extracted and transported retrogradely to a new cis cisterna behind. Likewise, the
medial enzymes would be received by retrograde transport from the cisternae just ahead. In this way, a
cis cisterna would mature to a medial and then trans cisterna as it moves outward. (B) In the vesicle
transport model, Golgi cisternae are static compartments, which contain a characteristic complement of
resident enzymes. The passing of molecules from cis to trans through the Golgi is accomplished by
forwardmoving transport vesicles, which bud from one cisterna and fuse with the next in a cis-to-trans
direction. Figure 13–36 A model of golgin function. Filamentous golgins anchored to Golgi membranes
capture transport vesicles by binding to Rab proteins on the vesicle surface. MBoC6 n13.120/13.36 Rab
interactor domain Golgi stack golgins transport vesicle Rab protein cytoskeletal interactor domain
Cisternal Maturation Model Cisternae mature in composition as they move along microtubules Golgi
resident proteins that are carried forward in a maturing cisterna are retrieved to an earlier compartment
in COPIcoated vesicles. When a newly formed cisterna moves to a medial position, for example,
“leftover” cis golgi enzymes would be extracted and transported retrogradely to a new cis cisterna
behind. Likewise, the medial enzymes would be received by retrograde transport from the cisternae just
ahead. In this way, a cis cisterna would mature to a medial and then trans cisterna as it moves outward.
Two Models for Compartmentalisation Vesicle Transport Model Cisternae are static compartments,
which contain a characteristic set of resident proteins and enzymes. The passing of molecules from cis to
trans through the golgi is accomplished by forward- moving transport vesicles, which bud from one
cisterna and fuse with the next in a cis-to-trans direction. 721 retrieves escaped ER and Golgi proteins
and returns them to upstream compartments. Directional fow could be achieved because forward-
moving cargo molecules are selectively packaged into forward-moving vesicles. Although both forward-
and backward-moving vesicles would likely be COPI-coated, the coats may contain diferent adaptor
proteins that confer selectivity on the packaging of cargo molecules. Alternatively, transport vesicles
shuttling between Golgi cisternae might not be directional at all, transporting cargo randomly back and
forth; directional fow would then occur because of the continual input to the cis cisterna and output
from the trans cisterna. Te vesicle transport model is supported by experiments that show that cargo
molecules are present in small COPI-coated vesicles and that these vesicles can deliver them to Golgi
cisternae over large distances. In addition, when experimentally aggregated membrane proteins are
introduced into Golgi cisternae, they can be observed staying in place, while soluble cargo, even if
present as large aggregates, traverses the Golgi at normal rates. It is likely that aspects of both models
are true. A stable core of long-lasting cisternae might exist in the center of each Golgi cisterna, while
regions at the rim may undergo continuous maturation, perhaps utilizing Rab cascades that change their
identity. As matured pieces of the cisternae are formed, they might break of and fuse with downstream
cisternae by homotypic fusion mechanisms, taking large cargo molecules with them. In addition, small
COPI-coated vesicles might transport small cargo in the forward direction and retrieve escaped Golgi
enzymes and return them to their appropriate upstream cisternae. Golgi Matrix Proteins Help Organize
the Stack Te unique architecture of the Golgi apparatus depends on both the microtubule cytoskeleton,
as already mentioned, and cytoplasmic Golgi matrix proteins, which form a scafold between adjacent
cisternae and give the Golgi stack its structural integrity. Some of the matrix proteins, called golgins,
form long tethers composed of stif coiled-coil domains with interspersed hinge regions. Golgins form a
forest of tentacles that can extend 100–400 nm from the surface of the Golgi stack. Tey are thought to
help retain Golgi transport vesicles close to the organelle through interactions with Rab proteins (Figure
13–36). When the cell prepares to divide, mitotic protein kinases phosphorylate the Golgi matrix
proteins, causing the Golgi TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS (A) CISTERNAL
MATURATION MODEL (B) VESICLE TRANSPORT MODEL ER CGN TGN cisternae cis trans medial vesicular
tubular cluster MBoC6 m13.35/13.35 matrix proteins Figure 13–35 Two possible models explaining the
organization of the Golgi apparatus and how proteins move through it. It is likely that the transport
through the Golgi apparatus in the forward direction (red arrows) involves elements of both models. (A)
According to the cisternal maturation model, each Golgi cisterna matures as it migrates outward
through the stack. At each stage, the Golgi resident proteins that are carried forward in a maturing
cisterna are moved backward to an earlier compartment in COPI-coated vesicles. When a newly formed
cisterna moves to a medial position, for example, “leftover” cis Golgi enzymes would be extracted and
transported retrogradely to a new cis cisterna behind. Likewise, the medial enzymes would be received
by retrograde transport from the cisternae just ahead. In this way, a cis cisterna would mature to a
medial and then trans cisterna as it moves outward. (B) In the vesicle transport model, Golgi cisternae
are static compartments, which contain a characteristic complement of resident enzymes. The passing
of molecules from cis to trans through the Golgi is accomplished by forwardmoving transport vesicles,
which bud from one cisterna and fuse with the next in a cis-to-trans direction. Figure 13–36 A model of
golgin function. Filamentous golgins anchored to Golgi membranes capture transport vesicles by binding
to Rab proteins on the vesicle surface. MBoC6 n13.120/13.36 Rab interactor domain Golgi stack golgins
transport vesicle Rab protein cytoskeletal interactor domain 714 Chapter 13: Intracellular Membrane
Traffic Unlike the retrieval signals on ER membrane proteins, which can interact directly with the COPI
coat, soluble ER resident proteins must bind to specialized receptor proteins such as the KDEL receptor
—a multipass transmembrane protein that binds to the KDEL sequence and packages any protein
displaying it into COPI-coated retrograde transport vesicles (Figure 13–25). To accomplish this task, the
KDEL receptor itself must cycle between the ER and the Golgi apparatus, and its afnity for the KDEL
sequence must difer in these two compartments. Te receptor must have a high afnity for the KDEL
sequence in vesicular tubular clusters and the Golgi apparatus, so as to capture escaped, soluble ER
resident proteins that are present there at low concentration. It must have a low afnity for the KDEL
sequence in the ER, however, to unload its cargo in spite of the very high concentration of KDEL-
containing soluble resident proteins in the ER. How does the afnity of the KDEL receptor change
depending on the compartment in which it resides? Te answer is likely related to the lower pH in the
Golgi compartments, which is regulated by H+ pumps. As we discuss later, pH-sensitive protein–protein
interactions form the basis for many of the protein sorting steps in the cell. Most membrane proteins
that function at the interface between the ER and Golgi apparatus, including v- and t-SNAREs and some
cargo receptors, also enter the retrieval pathway back to the ER. Many Proteins Are Selectively Retained
in the Compartments in Which They Function Te KDEL retrieval pathway only partly explains how ER
resident proteins are maintained in the ER. As mentioned, cells that express genetically modifed ER
resident proteins, from which the KDEL sequence has been experimentally removed, secrete these
proteins. But the rate of secretion is much slower than for a normal secretory protein. It seems that a
mechanism that is independent of their KDEL signal normally retains ER resident proteins and that only
those proteins that escape this retention mechanism are captured and returned via the KDEL receptor. A
suggested retention mechanism is that ER resident proteins bind to one another, thus forming
complexes that are too big to enter transport vesicles efciently. Because ER resident proteins are
present in the ER at very high concentrations (estimated to be millimolar), relatively low-afnity
interactions would sufce to retain most of the proteins in such complexes. Aggregation of proteins that
function in the same compartment is a general mechanism that compartments use to organize and
retain their resident proteins. Golgi enzymes that function together, for example, also bind to each
other and are thereby restrained from entering transport vesicles leaving the Golgi apparatus. Figure
13–25 Retrieval of soluble ER resident proteins. ER resident proteins that escape from the ER are
returned by vesicle transport. (A) The KDEL receptor present in both vesicular tubular clusters and the
Golgi apparatus captures the soluble ER resident proteins and carries them in COPI-coated transport
vesicles back to the ER. (Recall that the COPIcoated vesicles shed their coats as soon as they are
formed.) Upon binding its ligands in the tubular cluster or Golgi, the KDEL receptor may change
conformation, so as to facilitate its recruitment into budding COPI-coated vesicles. (B) The retrieval of ER
proteins begins in vesicular tubular clusters and continues from later parts of the Golgi apparatus. In the
environment of the ER, the ER resident proteins dissociate from the KDEL receptor, which is then
returned to the Golgi apparatus for reuse. We discuss the different compartments of the Golgi
apparatus shortly. soluble ER resident protein vesicular tubular cluster or Golgi apparatus KDEL empty
KDEL receptor COPI coat FORWARD PATHWAY RETRIEVAL PATHWAY secretory protein COPII coat COPI
coat KDEL receptor protein soluble ER resident protein ER vesicular tubular cluster cis Golgi network cis,
medial, and trans Golgi cisternae trans Golgi network (A) (B) MBoC6 m13.24/13.24 Retrieval of soluble
ER resident proteins ER resident proteins that escape from the ER are returned by vesicle transport The
KDEL receptor present in both vesicular tubular clusters and the golgi apparatus captures the soluble ER
resident proteins and carries them in COPI-coated transport vesicles back to the ER How do proteins sort
between the ER and the Golgi apparatus? 717 Two broad classes of N-linked oligosaccharides, the
complex oligosaccharides and the high-mannose oligosaccharides, are attached to mammalian
glycoproteins. Sometimes, both types are attached (in diferent places) to the same polypeptide chain.
Complex oligosaccharides are generated when the original N-linked oligosaccharide added in the ER is
trimmed and further sugars are added; by contrast, high-mannose oligosaccharides are trimmed but
have no new sugars added to them in the Golgi apparatus (Figure 13–30). Te sialic acids in the
TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS Golgi apparatus plasma membrane cis
cisterna medial cisterna trans cisterna Golgi stack trans Golgi network cis Golgi network ER lysosome
secretory vesicle • removal of Man • removal of Man • addition of GlcNAc • addition of Gal • addition of
NANA • sulfation of tyrosines and carbohydrates • phosphorylation of oligosaccharides on lysosomal
proteins SORTING SORTING MBoC6 m13.28/13.28 Figure 13–29 Oligosaccharide processing in Golgi
compartments. The localization of each processing step shown was determined by a combination of
techniques, including biochemical subfractionation of the Golgi apparatus membranes and electron
microscopy after staining with antibodies specific for some of the processing enzymes. Processing
enzymes are not restricted to a particular cisterna; instead, their distribution is graded across the stack,
such that early-acting enzymes are present mostly in the cis Golgi cisternae and later-acting enzymes are
mostly in the trans Golgi cisternae. Man, mannose; GlcNAc, N-acetylglucosamine; Gal, galactose; NANA,
N-acetylneuraminic acid (sialic acid). Figure 13–30 The two main classes of asparagine-linked (N-linked)
oligosaccharides found in mature mammalian glycoproteins. (A) Both complex oligosaccharides and
high-mannose oligosaccharides share a common core region derived from the original N-linked
oligosaccharide added in the ER (see Figure 12–50) and typically containing two N-acetylglucosamines
(GlcNAc) and three mannoses (Man). (B) Each complex oligosaccharide consists of a core region,
together with a terminal region that contains a variable number of copies of a special trisaccharide unit
(N-acetylglucosamine–galactose–sialic acid) linked to the core mannoses. Frequently, the terminal
region is truncated and contains only GlcNAc and galactose (Gal) or just GlcNAc. In addition, a fucose
may be added, usually to the core GlcNAc attached to the asparagine (Asn). Thus, although the steps of
processing and subsequent sugar addition are rigidly ordered, complex oligosaccharides can be
heterogeneous. Moreover, although the complex oligosaccharide shown has three terminal branches,
two and four branches are also common, depending on the glycoprotein and the cell in which it is made.
(C) High-mannose oligosaccharides are not trimmed back all the way to the core region and contain
additional mannoses. Hybrid oligosaccharides with one Man branch and one GlcNAc and Gal branch are
also found (not shown). The three amino acids indicated in (A) constitute the sequence recognized by
the oligosaccharyl transferase enzyme that adds the initial oligosaccharide to the protein. Ser, serine;
Thr, threonine; X, any amino acid, except proline. Asn X Ser or Thr NH CO = N-acetylglucosamine
(GlcNAc) = mannose (Man) = galactose (Gal) = N-acetylneuraminic acid (sialic acid, or NANA) KEY (A) (B)
COMPLEX OLIGOSACCHARIDE (C) HIGH-MANNOSE OLIGOSACCHARIDE MBoC6 m13.30/13.30 CORE
REGION The localization of each processing step shown was determined by a combination of techniques,
including biochemical subfractionation of the golgi apparatus membranes and electron microscopy after
staining with antibodies specific for some of the processing enzymes. processing enzymes are not
restricted to a particular cisterna; instead, their distribution is graded across the stack, such that early-
acting enzymes are present mostly in the cis golgi cisternae and later-acting enzymes are mostly in the
trans golgi cisternae. Man - Mannose GlcNac - N-Acetylglucosamine Gal - Galactose NANA - N-
Acetylneuraminic acid (sialic acid) Golgi apparatus functions in glycosylation 719 or, in some cases—such
as collagens—to hydroxylated proline and lysine side chains. Tis O-linked glycosylation (Figure 13–32),
like the extension of N-linked oligosaccharide chains, is catalyzed by a series of glycosyl transferase
enzymes that use the sugar nucleotides in the lumen of the Golgi apparatus to add sugars to a protein
one at a time. Usually, N-acetylgalactosamine is added frst, followed by a variable number of additional
sugars, ranging from just a few to 10 or more. Te Golgi apparatus confers the heaviest O-linked
glycosylation of all on mucins, the glycoproteins in mucus secretions, and on proteoglycan core proteins,
which it modifes to produce proteoglycans. As discussed in Chapter 19, this process involves the
polymerization of one or more glycosaminoglycan chains (long, unbranched polymers composed of
repeating disaccharide units; see Figure 19–35) onto serines on a core protein. Many proteoglycans are
secreted and become components of the extracellular matrix, while others remain anchored to the
extracellular face of the plasma membrane. Still others form a major component of slimy materials, such
as the mucus that is secreted to form a protective coating on the surface of many epithelia. Te sugars
incorporated into glycosaminoglycans are heavily sulfated in the Golgi apparatus immediately after
these polymers are made, thus adding a signifcant portion of their characteristically large negative
charge. Some tyrosines in proteins also become sulfated shortly before they exit from the Golgi
apparatus. In both cases, the sulfation depends on the sulfate donor 3ʹ-phosphoadenosine-5ʹ-
phosphosulfate (PAPS) (Figure 13–33), which is transported from the cytosol into the lumen of the trans
Golgi network. What Is the Purpose of Glycosylation? Tere is an important diference between the
construction of an oligosaccharide and the synthesis of other macromolecules such as DNA, RNA, and
protein. Whereas nucleic acids and proteins are copied from a template in a repeated series of identical
steps using the same enzyme or set of enzymes, complex carbohydrates require a diferent enzyme at
each step, each product being recognized as the exclusive substrate for the next enzyme in the series. Te
vast abundance of glycoproteins and the complicated pathways that have evolved to synthesize them
emphasize that the oligosaccharides on glycoproteins and glycosphingolipids have very important
functions. N-linked glycosylation, for example, is prevalent in all eukaryotes, including yeasts. N-linked
oligosaccharides also occur in a very similar form in archaeal cell wall proteins, suggesting that the
whole machinery required for their synthesis is evolutionarily ancient. N-linked glycosylation promotes
protein folding in two ways. First, it has a direct role in making folding intermediates more soluble,
thereby preventing their aggregation. Second, the sequential modifcations of the N-linked
oligosaccharide establish a “glyco-code” that marks the progression of TRANSPORT FROM THE ER
THROUGH THE GOLGI APPARATUS C H H H H NHCOCH3 HOH2C HOH CH2 C O O O NH protein backbone
Asparagine N-acetylglucosamine remainder of oligosaccharide side chain N-LINKED GLYCOSYLATION C H
H H NHCOCH3 H H OH 3C CHH CH2OH O O protein backbone Threonine N-acetylgalactosamine
remainder of oligosaccharide side chain O-LINKED GLYCOSYLATION O MBoC6 m13.32/13.32 Figure 13–
32 N- and O-linked glycosylation. In each case, only the single sugar group that is directly attached to the
protein chain is shown. H H H H HO O O H N N N N NH2 O CH2 P P O O O OO S O– O– O– –O 3′-
phosphoadenosine-5′-phosphosulfate (PAPS) MBoC6 m13.33/13.33 Figure 13–33 The structure of PAPS.
Two types of glycosylation 718 Chapter 13: Intracellular Membrane Traffic complex oligosaccharides are
of special importance because they bear a negative charge. Whether a given oligosaccharide remains
high-mannose or is processed depends largely on its position in the protein. If the oligosaccharide is
accessible to the processing enzymes in the Golgi apparatus, it is likely to be converted to a complex
form; if it is inaccessible because its sugars are tightly held to the protein’s surface, it is likely to remain
in a high-mannose form. Te processing that generates complex oligosaccharide chains follows the highly
ordered pathway shown in Figure 13–31. Beyond these commonalities in oligosaccharide processing
that are shared among most cells, the products of the carbohydrate modifcations carried out in the
Golgi apparatus are highly complex and have given rise to a new feld of study called glycobiology. Te
human genome, for example, encodes hundreds of different Golgi glycosyl transferases and many
glycosidases, which are expressed differently from one cell type to another, resulting in a variety of
glycosylated forms of a given protein or lipid in diferent cell types and at varying stages of diferentiation,
depending on the spectrum of enzymes expressed by the cell. Te complexity of modifcations is not
limited to N-linked oligosaccharides but also occurs on O-linked sugars, as we discuss next.
Proteoglycans Are Assembled in the Golgi Apparatus In addition to the N-linked oligosaccharide
alterations made to proteins as they pass through the Golgi cisternae en route from the ER to their fnal
destinations, many proteins are also modifed in the Golgi apparatus in other ways. Some proteins have
sugars added to the hydroxyl groups of selected serines or threonines, Asn glucosidase II glucosidase I
ER mannosidase Asn Golgi mannosidase I Asn UDP UDP Asn Golgi mannosidase II Asn Asn 1 2 3 4 5 ER
LUMEN Endo Hsensitive Endo Hresistant next added here N-acetylglucosamine transferase I = N-
acetylglucosamine (GlcNAc) = mannose (Man) = glucose (Glc) = galactose (Gal) = N-acetylneuraminic acid
(sialic acid, or NANA) MBoC6 m13.31/13.31 CMP3 5 + UDP 3 CMP UDP3 UDP2 GOLGI LUMEN complex
oligosaccharide high-mannose oligosaccharide KEY: Figure 13–31 Oligosaccharide processing in the ER
and the Golgi apparatus. The processing pathway is highly ordered, so that each step shown depends on
the previous one. Step 1: Processing begins in the ER with the removal of the glucoses from the
oligosaccharide initially transferred to the protein. Then a mannosidase in the ER membrane removes a
specific mannose. The remaining steps occur in the Golgi stack. Step 2: Golgi mannosidase I removes
three more mannoses. Step 3: N-acetylglucosamine transferase I then adds an N-acetylglucosamine.
Step 4: Mannosidase II then removes two additional mannoses. This yields the final core of three
mannoses that is present in a complex oligosaccharide. At this stage, the bond between the two N-
acetylglucosamines in the core becomes resistant to attack by a highly specific endoglycosidase (Endo
H). Since all later structures in the pathway are also Endo H-resistant, treatment with this enzyme is
widely used to distinguish complex from high-mannose oligosaccharides. Step 5: Finally, as shown in
Figure 13–30, additional N-acetylglucosamines, galactoses, and sialic acids are added. These final steps
in the synthesis of a complex oligosaccharide occur in the cisternal compartments of the Golgi
apparatus: three types of glycosyl transferase enzymes act sequentially, using sugar substrates that have
been activated by linkage to the indicated nucleotide; the membranes of the Golgi cisternae contain
specific carrier proteins that allow each sugar nucleotide to enter in exchange for the nucleoside
phosphates that are released after the sugar is attached to the protein on the lumenal face. Note that,
as a biosynthetic organelle, the Golgi apparatus differs from the ER: all sugars in the Golgi are assembled
inside the lumen from sugar nucleotide, whereas in the ER, the N-linked precursor oligosaccharide is
assembled partly in the cytosol and partly in the lumen, and most lumenal reactions use dolichol-linked
sugars as their substrates (see Figure 12–51). Oligosaccharide processing in the ER and the Golgi
apparatus. The processing pathway is highly ordered, so that each step shown depends on the previous
one. Step 1: processing begins in the ER with the removal of the glucose residues from the
oligosaccharide initially transferred to the protein. Then a mannosidase in the ER membrane removes a
specific mannose. The remaining steps occur in the Golgi stack. Step 2: Golgi mannosidase I removes
three more mannose residues. Step 3: N-Acetylglucosamine transferase I then adds an N-
Acetylglucosamine. Step 4: Mannosidase II then removes two additional mannose residues. This yields
the final core of three mannose residues that is present in a complex oligosaccharide. Step 5: Additional
N-Acetylglucosamines, galactoses, and sialic acids are added. These final steps in the synthesis of a
complex oligosaccharide occur in the cisternal compartments of the Golgi apparatus. Three types of
glycosyl transferase enzymes act sequentially, using sugar substrates that have been activated by linkage
to the indicated nucleotide; the membranes of the Golgi cisternae contain specific carrier proteins that
allow each sugar nucleotide to enter in exchange for the nucleoside phosphates that are released after
the sugar is attached to the protein on the lumenal face.