Angewandte
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DOI: 10.1002/anie.201210178
Protein–Lipid Interactions
In Vivo Profiling and Visualization of Cellular Protein–Lipid
Interactions Using Bifunctional Fatty Acids**
Per Haberkant,* Reinout Raijmakers, Marjolein Wildwater, Timo Sachsenheimer, Britta Brügger,
Kenji Maeda, Martin Houweling, Anne-Claude Gavin, Carsten Schultz, Gerrit van Meer,
Albert J. R. Heck, and Joost C. M. Holthuis
Cellular processes are mediated by the concerted action of
numerous biomolecules that form complex interaction networks. Considerable efforts have been devoted to elucidating
the cellular interactome, with the majority of studies focusing
on mapping protein–protein, protein–DNA, and protein–
metabolite interaction networks.[1, 2] Yet two-thirds of the
cellular proteome operates at a membrane surface or within
a membrane comprising thousands of different lipid species.
Besides serving as essential building blocks of membranes
and anhydrous stores of energy, lipids participate in a multitude of signaling pathways. Perturbations in lipid homeostasis
frequently result in human diseases, ranging from neurodegenerative disorders to metabolic syndrome and cancer.[3, 4]
While these findings imply an intricate interplay between
proteins and lipids, only a few studies have been carried out to
chart protein–lipid interactions in a systematic fashion.
To catalogue protein–lipid interactions in yeast, Gallego
et al. used a comprehensive set of tandem affinity-purified
proteins and probed these on miniaturized nitrocellulose lipid
arrays; this work led to the novel concept that pleckstrin
homology domains can have dual binding specificities and
may act as coincidence sensors for distinct lipid classes.[5]
Other studies focused on the application of radioactive
photoactivatable lipids to investigate individual protein–
lipid interactions in living cells.[6, 7] This approach recently
led to the identification of a preferential interaction of the
membrane protein p24 with a single species of sphingomyelin,
with the lipid serving as a cofactor in the formation of coat
protein I (COPI) vesicles.[8] In addition, phospholipid-based
Dr. M. Houweling
Department of Biochemistry and Cell Biology
Faculty of Veterinary Medicine
P.O. Box 80176, 3508 TD Utrecht (The Netherlands)
[*] Dr. P. Haberkant, Dr. K. Maeda, Dr. A.-C. Gavin,
Priv.-Doz. Dr. C. Schultz
European Molecular Biology Laboratory
Meyerhofstrasse 1, 69117 Heidelberg (Germany)
E-mail: per.haberkant@embl.de
Dr. P. Haberkant, Dr. R. Raijmakers, Dr. M. Wildwater,
Prof. G. van Meer, Prof. A. J. R. Heck, Prof. J. C. M. Holthuis
Membrane Biochemistry & Biophysics, Biomolecular Mass Spectrometry & Proteomics Group, Developmental Biology, Bijvoet
Center for Biomolecular Research
Utrecht University
3584 CH Utrecht (The Netherlands)
Prof. J. C. M. Holthuis
Molecular Cell Biology, University of Osnabrck
Barbarastrasse 13, 49076 Osnabrck (Germany)
T. Sachsenheimer, Priv.-Doz. Dr. B. Brgger
Heidelberg University Biochemistry Center
Im Neuenheimer Feld 328, 69120 Heidelberg (Germany)
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activity probes have been applied to characterize lipidbinding proteins using synthetic analogues of phosphatidylcholine and phosphatidylinositol polyphosphates.[9–11] Apart
from a photoaffinity moiety for the covalent labeling of target
proteins, these probes contain a second tag for click chemistry
to enable the detection and enrichment of the crosslinked
proteins for subsequent identification by mass spectrometry.
However, a major disadvantage of these bifunctional phospholipid analogues is that their application is restricted to cell
extracts, thus preventing a systematic analysis of protein–lipid
interactions in their native environment.
For a global analysis of protein–lipid interactions in living
cells, we have now synthesized a 15 carbon long photoactivatable and clickable fatty acid, pacFA (Figure 1 a). In the
initial step, the diprotected keto fatty acid 3 was obtained
from the Grignard reaction of the trimethylsilyl(TMS)protected alkyne 1 and acid chloride 2. In the second step,
the ester and TMS group were cleaved simultaneously under
alkaline conditions yielding the keto fatty acid 4. Conversion
of the keto group into a photoactivatable diazirine ring was
achieved as described previously,[6] yielding pacFA 5.
To screen for protein–lipid interactions in vivo, cells were
fed pacFA as a precursor for the biosynthesis of bifunctional
lipids. Proteins in direct contact with a bifunctional lipid were
then crosslinked by irradiation of the diazirine group with UV
light (Figure 1 b). This resulted in a highly reactive intermediate that, within the nanosecond range,[12] formed a covalent linkage to molecules in close proximity. Next, click
chemistry was used to label the alkyne group with a reporter
[**] This work was supported by the Marie Curie Actions COFUND
(EMBL Interdisciplinary Postdoc (EIPOD) stipend to P.H.), the
Netherlands Proteomics Center (NPC) within the Netherlands
Genomics Initiative and an NPC Valorization Voucher (P.H.), the
European Union (7th FP Marie-Curie ITN “SPHINGONET” to C.S.,
A.J.R.H., J.C.M.H.), and the DFG (TRR83 to B.B., C.S.). K.M. was
a fellow of the Danish Natural Science Research Council (09064986/FNU). We thank Y. Horibata, H. Sugimoto, and S. Cockcroft
for providing plasmids, C. Versluis for high-resolution mass
spectrometra, and H. Vlieg, H. Dijkstra, and T. Klimeck for technical
assistance.
Supporting information for this article (including detailed experimental procedures) is available on the WWW under http://dx.doi.
org/10.1002/anie.201210178.
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Figure 1. a) Synthesis of the bifunctional fatty acid pacFA 5. b) General
scheme for the in vivo profiling of cellular protein–lipid interactions.
pacFA fed to cells is metabolically incorporated into a bifunctional
lipid (marked by asterisk), which can be crosslinked to its proteinbinding partner (green) by UV irradiation. Click chemistry is used to
label the alkyne group in the bifunctional lipid with biotin-N3 or
fluorescein-N3 (Fluo-N3), allowing the respective identification or
visualization of the crosslinked protein–lipid complex.
molecule, allowing the visualization or affinity-purification of
the crosslinked products. An important advantage of this twostep procedure is that the functionalized lipid is tagged after
its interaction with a protein-binding partner has been
captured by UV-crosslinking, so that the bulky reporter
molecule does not interfere with the protein–lipid interaction.
As a proof-of-principle experiment, pacFA was incubated
with the fatty acid carrier protein bovine serum albumin
(BSA) and subjected to UV irradiation. Next, the mixture was
subjected to a click reaction with biotin azide and analyzed by
immunoblotting. Tagging of BSA with biotin was observed
only after UV irradiation and in the presence of pacFA
(Figure S1 in the Supporting Information). Biotinylation of
BSA was strictly dependent on CuSO4 and the reducing agent
tris(2-carboxyethyl)phosphine (TCEP), two essential components of the click reaction. No photoaffinity labeling was
observed when BSA was replaced with lysozyme, a protein
lacking a known fatty acid binding site. Thus, photoaffinity
labeling of BSA relies on a specific interaction between the
protein and pacFA.
To investigate whether pacFA is a suitable precursor for
the biosynthesis of phospholipids, we fed pacFA to Chinese
hamster ovary (CHO) and human HeLa cells for 16 h and
analyzed cellular lipid extracts for the presence of bifunctional species of phosphatidylcholine (PC) using nano-electrospray ionization tandem mass spectrometry (nano-ESIMS/MS). The labeling of CHO cells with pacFA yielded peaks
that showed a 8 Da increase in the m/z ratio relative to the
peaks of endogenous PC species, a shift corresponding to the
difference in the molecular weights of pacFA (264 g mol 1)
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and its natural analogue, palmitic acid (256 g mol 1; Figure S2
in the Supporting Information). Multiple precursor ion scans
(MPIS) of lipid extracts from pacFA-fed human HeLa cells
for the characteristic pacFA ion ([M H] , m/z 263) revealed
the presence of various pacFA-containing PC species (Figure S3 a in the Supporting Information). pacFA was also
incorporated into phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL) (Figures S3 b and S4 in the
Supporting Information).
To monitor the metabolic turnover of pacFA, lipid
extracts of pacFA-fed HeLa cells were subjected to a click
reaction with 3-azido-7-hydroxycoumarin and then analyzed
by thin-layer chromatography as described for other alkynylated lipids.[13] Within 1 h of metabolic labeling, the bulk of
pacFA (> 95 %) was incorporated into diacylglycerol (DAG,
16 %), triacylglycerol/cholesterol ester (TAG/CE, 17 %), and
phospholipids (49 %; Figure S5 in the Supporting Information). Increasing the labeling time to 16 h or substituting
pacFA for palmitic acid alkyne, a clickable fatty acid (cFA),
resulted in a very similar distribution of the label over these
major lipid classes. Thus, pacFA is readily converted into
a variety of bifunctional lipids including all major classes of
phospholipids. In contrast, we found no evidence for the
incorporation of pacFA into sphingolipids, a somewhat surprising result as palmitic acid is a precursor for the production
of sphinganine and ceramide by serine palmitoyltransferase
and ceramide synthase, respectively. As both enzymes display
a high level of specificity towards acyl chain length,[14–16] the
introduction of the diarizine and alkyne groups may cause
a length mismatch that prevents the recognition of pacFA as
a substrate for sphingolipid biosynthesis.
We next investigated the application of pacFA as a tool for
capturing protein–lipid interactions in living cells. To this end,
CHO cells were fed pacFA for 1 h or 16 h followed by UV
irradiation and cell lysis. The cell membranes were separated
from the cytosol and subjected to a click reaction with
Alexa 488 azide. Alexa-labeled proteins were visualized by
SDS-PAGE and in-gel fluorescence. As shown in Figure S6 in
the Supporting Information, a subset of membrane proteins in
pacFA-fed, UV-irradiated cells was fluorescence-labeled
whereas cytosolic proteins were essentially devoid of labeling.
In the absence of UV irradiation, a subset of membrane
proteins was labeled, but only in cells fed with pacFA for 16 h
(Figure 2 a). No labeling was observed when pacFA was
omitted. Thus, in cells fed with pacFA for 16 h, some
membrane proteins undergo fluorescence labeling irrespective of photo-crosslinking. As a small fraction of cellular
proteins is modified by palmitoylation, the labeling of
proteins in non-UV-irradiated cells could be due to the
metabolic incorporation of pacFA. Consistent with this
notion, UV-independent labeling of proteins was also
observed in cFA-fed cells (Figure S7 in the Supporting
Information).[17] To address this further, total membranes
from pacFA-fed non-UV-irradiated CHO cells were subjected to a click reaction with biotin azide. Click reactions on
membranes from cells grown in the absence of pacFA served
as a control. Next, biotin-derivatized proteins were visualized
by immunoblotting and affinity-purified using Neutravidin
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Figure 2. Global profiling of protein–lipid interactions in pacFA-fed CHO cells. a) CHO cells were grown for 1 h or 16 h in the presence or absence
of pacFA and then subjected to UV irradiation, as indicated. Total cellular membranes were isolated, subjected to a click reaction using Alexa 488
azide, processed for SDS-PAGE, and then analyzed by in-gel fluorescence (left panel) or stained with Coomassie brilliant blue (right panel).
b) Total membranes from CHO cells treated as in (a) were subjected to a click reaction using biotin azide, processed for SDS-PAGE, and then
subjected to Western blotting using an anti-biotin antibody. c) Biotinylated protein–lipid complexes were purified from total cellular membranes
using Neutravidin beads and then subjected to SDS-PAGE and staining with Coomassie brillant blue. Note that the prominent bands at
approximately 15 kDa correspond to monomeric subunits of Neutravidin. d) Mass spectrometric identification of proteins affinity-purified with
Neutravidin from pacFA-fed CHO cells prior to (left panel) or after UV irradiation (right panel). Presented are the numbers of proteins that were
identified from two independent experiments. e) Subcellular distribution of the proteins identified in (d). f) Clustering of functions assigned to
proteins affinity-purified from pacFA-fed, UV-irradiated CHO cells that were identified twice in two independent experiments.
beads (Figure 2 b,c). The purified proteins were separated by
SDS-PAGE, subjected to in-gel tryptic digestion and identified by mass spectrometry (criteria for the identification of
crosslinked proteins are defined in the Supporting Information). Two independent experiments yielded a total of 185
proteins that were exclusively present in affinity-purified
fractions of pacFA-fed non-UV-irradiated cells, of which 64
proteins were identified twice (Figure 2 d, Table S2 in the
Supporting Information). About 6.5 % of these proteins (12
out of 185) are known targets of palmitoylation, whereas 26 %
(48 out of 185) correspond to proteins identified previously in
a global screen for palmitoylated proteins.[18] Moreover, about
half of the 185 proteins are predicted to localize in compartments where most palmitolyated proteins are known to occur
(i.e. cytosol, nucleus, plasma membrane; Figure 2 e).[19]
Together, these results indicate that the affinity-purified
proteins of non-UV-irradiated pacFA-fed cells are enriched
in palmitoylated proteins and that pacFA is incorporated into
palmitoylated proteins.
To identify proteins that are crosslinked to bifunctional
lipids, the total membranes from pacFA-fed UV-irradiated
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CHO cells were reacted with biotin azide and the proteins
were affinity-purified using Neutravidin beads prior to mass
spectrometric analysis. Non-UV-irradiated pacFA-fed cells
served as a control to distinguish lipid-crosslinked proteins
from palmitoylated proteins. Two independent experiments
yielded a total of 253 proteins that were either exclusively
present or highly enriched in affinity-purified fractions of
pacFA-fed UV-irradiated cells. Nearly 40 % of these lipidcrosslinked proteins (96 out of 253) were identified twice
(Figure 2 d; Table S3 in the Supporting Information). In
contrast to the proteins that were affinity-purified from
non-UV-irradiated cells, the bulk of the lipid-crosslinked
proteins isolated from UV-irradiated cells corresponded to
endoplasmic reticulum (ER) resident or mitochondrial proteins (Figure 2 e). These proteins participate in a wide range
of cellular processes, including lipid metabolism, protein
biosynthesis, membrane trafficking, transport, and signaling
(Figure 2 f).
To further validate our findings, we identified lipidcrosslinked proteins from pacFA-fed, UV-irradiated HeLa
cells (Figure S8 in the Supporting Information). In one
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experiment, HeLa cells were fed pacFA for 16 h prior to UV
irradiation. In a second experiment, the pacFA labeling time
was shortened to 1 h. Omission of UV irradiation served as
a control to discriminate lipid-crosslinked proteins from
palmitoylated proteins, as described above. As shown in
Figure 3 a, the number of putative palmitoylated proteins
Figure 3. Imaging of protein–lipid interactions in pacFA-fed HeLa cells.
a) Mass spectrometric identification of proteins affinity-purified with
Neutravidin from pacFA-fed HeLa cells prior to (left panel) or after UV
irradiation (right panel). Presented are the numbers of proteins that
were identified from two independent experiments on cells that were
fed pacFA for 1 h or 16 h. b) HeLa cells stably expressing RFP-tagged
Golgi-resident mannisodase II were fed pacFA for 1 h and UV-irradiated, as indicated. Cells were fixed in methanol, extracted with chloroform/methanol/acetic acid, subjected to a click reaction with Alexa 488
azide, and then visualized by fluorescence microscopy. Bottom row:
magnification of regions as indicated in the middle panel. Bars,
10 mm.
identified in non-UV-irradiated cells dropped by more than
fourfold when cells were fed pacFA for 1 h instead of 16 h
(from 214 to 49; see also Table S4 in the Supporting
Information). In contrast, the number of lipid-crosslinked
proteins identified in UV-irradiated cells was largely independent of whether cells were fed pacFA for 1 h or 16 h (267
and 202, respectively; Figure 3 a and Table S5 in the Supporting Information). The bulk of these lipid-crosslinked proteins
were identified twice (176 out of 293), in line with our finding
that cells fed pacFA for 1 h or 16 h contain very similar
collections of bifunctional lipids (Figure S5 in the Supporting
Information). There was also a substantial overlap between
lipid-crosslinked proteins identified in HeLa and CHO cells.
In HeLa cells we found orthologs for 105 of the 253 lipidcrosslinked proteins isolated from CHO cells (42 %; Figure S8 c in the Supporting Information). Table S1 in the
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Supporting Information lists the top 99 high-confidence lipidcrosslinked proteins that were identified in both cell types and
at least three times out of four independent experiments.
Almost half of these proteins localize to the ER, whereas the
second largest fraction (nearly 20 %) comprises mitochondrial proteins. One-tenth of these proteins had previously
been shown to handle fatty acids or fatty acid derived
metabolites as their primary function. Among these were
proteins that catalyze fatty acid import and catabolism in
mitochondria (CPT2, ECHA), participate in phospholipid
biosynthesis (CDIPT), transport (PITPB, STARD7), and
turnover (ABHD6), mediate protein acylation in the ER
(PIGS, GPI8), or control long-chain base levels for sphingolipid biosynthesis in the ER (ORML2, SGPL1).
The identification of two cytosolic phospholipid-transfer
proteins, PITPB and STARD7, as high-confidence lipidcrosslinked proteins is of particular interest. To come in close
contact with the photoactivatable group, these proteins must
dip into the hydrophobic core of the bilayer or extract the
bifunctional lipid from the membrane surface. While the
precise cellular function of PITPB and STARD7 remains to
be established, both proteins contain specific phospholipid
binding sites and stimulate the monomeric transfer of
particular phospholipids between membranes in vitro.[20–22]
To gain further insight into the nature of the protein–lipid
interactions by which PITPB and STARD7 were captured in
our screen, the proteins were produced in E. coli and
incubated with liposomes prepared from lipid extracts of
HeLa cells that had been fed pacFA for 16 h. After the
removal of unbound lipids by size-exclusion chromatography,
the lipids bound to PITPB and STARD7 were identified by
TLC and mass spectrometry. Consistent with their known
lipid-binding specificities, PITPB interacted with both pacPC
and pacPI, whereas STARD7 interacted exclusively with
pacPC (Figures S9 and S10 in the Supporting Information).
The protein-bound lipid fractions were devoid of pacFA or
other bifunctional lipid species. Collectively, these data
demonstrate the suitability of pacFA as a tool to identify
protein–lipid interactions in living cells.
To test whether pacFA can also be used to visualize
cellular protein–lipid interactions in situ, HeLa cells were fed
pacFA for 1 h prior to UV irradiation. Omission of pacFA or
UV irradiation served as controls (Figure 3 b and Figure S11
in the Supporting Information). Next, the cells were fixed in
methanol at 20 8C to precipitate cellular proteins and then
extracted with chloroform to remove all lipids that were not
covalently bound to proteins. Following a click reaction with
Alexa 488 azide, the cells were analyzed by fluorescence
microscopy. To minimize the metabolic incorporation of
pacFA into palmitoylated proteins, labeling time was
restricted to 1 h. As shown in Figure 3 b, pacFA-fed UVirradiated cells displayed a prominent reticular fluorescent
staining of the cytoplasm that was highly reminiscent of the
ER. This staining extended to the nuclear envelope and was
abolished upon omission of either pacFA or UV irradiation.
These results are consistent with our finding that nearly half
of the high-confidence lipid-crosslinked proteins identified
from pacFA-fed UV-irradiated cells reside in the ER
(Table S1 in the Supporting Information).
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We next investigated whether pacFA can be used to map
protein–lipid interactions in a multicellular organism such as
the nematode C. elegans. L2 larvae were fed pacFA at 20 8C
prior to UV irradiation. Total lysates were reacted with
Alexa 488 azide and Alexa-labeled proteins were visualized
by SDS-PAGE and in-gel fluorescence (Figure 4 a). For the
of crosslinked proteins localize in the ER (Figure 2 e). Weaker
staining was observed in the hypodermis, whereas the gonads
were essentially devoid of staining. Staining of the hypodermis, muscle and intestinal cells was completely abolished in
pacFA-labeled worms when UV irradiation was omitted. The
precise reason for this tissue-specific labeling pattern is
unclear, but it deserves mention that intestinal epithelia and
muscle cells generally harbor active fatty acid uptake
systems.[23, 24]
In summary, we have established the bifunctional fatty
acid pacFA as a novel tool for the global profiling of protein–
lipid interactions in living cells. Our approach combines the
assets of previously reported photoactivatable and radioactive lipid precursors with click chemistry, thereby eliminating the drawbacks of radioactivity while providing novel
opportunities for visual screens aimed at understanding how
lipids function within their physiological context. The development of bifunctional precursors of sterol and sphingolipid
biosynthesis is currently under way to complement this work
and to enable a systematic profiling of proteins that bind
sterols and sphingolipids, which constitute the other two main
classes of membrane lipids. We show that the application of
pacFA can be extended from cultured cells to C. elegans, thus
providing a fresh starting point for a systematic investigation
of protein–lipid interactions in the context of animal physiology and development.
Received: December 20, 2012
Published online: February 28, 2013
Figure 4. Imaging of protein–lipid interactions in pacFA-fed C. elegans.
a) Wild-type L2 larvae were fed pacFA and then subjected to UV
irradiation, as indicated. Total cellular membranes were isolated,
subjected to a click reaction using Alexa 488 azide, processed for SDSPAGE, and then analyzed by in-gel fluorescence (left panel) or stained
with Coomassie brilliant blue (right panel). b) C. elegans expressing
a GFP-tagged muscle-specific transcription factor were fed pacFA and
then subjected to UV irradiation, as indicated. Larvae were fixed in
methanol and acetone, extracted with chloroform/methanol/acetic
acid, subjected to a click reaction with Alexa 594 azide, stained with
4’,6-diamidino-2-phenylindole (DAPI) and then visualized by fluorescence microscopy. Images were taken from the head (I), mid-region
(II), and tail (III), as schematically indicated. Left and middle panels
show Alexa 488 fluorescence, whereas the right panels display a merge
of Alexa 594, DAPI, and GFP fluorescence. Bars, 10 mm.
majority of these proteins, labeling was abolished when
pacFA or UV irradiation was omitted. To visualize protein–
lipid interactions in the entire organism, pacFA-fed and UVirradiated L2 larvae were fixed in methanol and acetone.
Non-crosslinked lipids were removed by chloroform extraction as described above and subjected to a click reaction with
Alexa 488 azide. As shown in Figure 4 b, pacFA-fed larvae
displayed a bright fluorescent staining that was dependent on
UV irradiation. The observed staining was not uniform and
particularly prominent in intestinal epithelial cells as well as
in muscle cells in the pharyngeal region. Both cell types
displayed a reticular staining of the cytoplasm resembling the
ER, as was also observed in pacFA-fed and UV-irradiated
HeLa cells, which is in line with our finding that the majority
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.
Keywords: cell imaging · click chemistry · lipids ·
photoaffinity labeling · proteins
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