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Blackwell Munksgaard
# 2007 The Authors
Journal compilation # 2007 Blackwell Publishing Ltd
doi: 10.1111/j.1600-0854.2007.00612.x
Rab6-interacting Protein 1 Links Rab6 and
Rab11 Function
Stéphanie Miserey-Lenkei1,2,†, Francxois
Waharte1,3,†, Annick Boulet1,2, Marie-Hélène
Cuif4, Danielle Tenza1,5, Amed El Marjou1,6,
Gracxa Raposo1,5, Jean Salamero1,2,3,
Laurent Héliot7, Bruno Goud1,2,* and
Solange Monier1,2,8,*
1
Institut Curie, Centre de Recherche, Paris, 75248 France
Molecular Mechanisms of Intracellular Transport,
UMR CNRS 144, Institut Curie, 26 rue d‘Ulm,
75248 Paris Cedex 05, France
3
Cell and Tissue Imaging Facility, UMR CNRS 144, Institut
Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France
4
UMR CNRS 8621, University of Paris 11, Orsay, France
5
Structure and Membrane Compartments, UMR CNRS
144, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05,
France
6
Recombinant Protein Facilities, UMR CNRS 144,
Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France
7
Biophotonic Group, IRI, Lille, France
8
Present address: UMR CNRS 6061, Université de
Rennes, 35043 Rennes, France
*Corresponding author: Bruno Goud, bruno.goud@curie.fr
or Solange Monier, solange.monier@curie.fr
†These authors contributed equally to this work.
2
Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs)
and Golgi complex, respectively. Evidence indicates that
they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the
existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1),
originally identified as a Rab6-binding protein. R6IP1 also
binds to Rab11A in its GTP-bound conformation. In
interphase cells, R6IP1 is targeted to the Golgi in
a Rab6-dependent manner but can associate with
Rab11-positive compartments when the level of Rab11A
is increased within the cells. Fluorescence resonance
energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an
interaction between Rab11A and Rab6 in living cells.
Accordingly, the REs marked by Rab11 and transferrin
receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal
and Golgi membranes do not appear to fuse with each
other. We also show that R6IP1 function is required
during metaphase and cytokinesis, two mitotic steps in
which a role of Rab6 and Rab11 has been previously
documented. We propose that R6IP1 may couple Rab6
and Rab11 function throughout the cell cycle.
Key words: cytokinesis, FRET/FLIM, Golgi complex, intracellular transport, mitosis, Rab GTPases, recycling endosomes
Received 16 February 2007, revised and accepted for publication 11 June 2007, uncorrected manuscript published
online 15 June 2007, published online 24 August 2007
Small guanosine triphosphatases (GTPases) of the Rab
family are recognized as key regulators of intracellular
transport and membrane trafficking within eukaryotic cells
(1). They function as molecular switches, cycling between
GDP- and GTP-bound states and between cytosol and
membranes. Through specific interactions with a wide
variety of effectors, Rab GTPases participate in the formation of transport intermediates, in their movement along
cytoskeletal tracks and in their docking/fusion with acceptor membranes. Rab proteins also play an important
role in the organization and dynamics of membrane
domains on organelles of the endocytic and exocytic
pathways (2,3).
The action of Rab GTPases needs to be co-ordinated in
order to ensure the transport directionality and to maintain
the identity of each intracellular compartment. Rab coupling
can be accomplished by proteins that are able to interact
with more than one Rab protein. Many examples of such
proteins now exist in the literature, although functional data
are still lacking for most of them. They can schematically be
divided into two main classes. The first comprises ‘bifunctional’ Rab effectors that bind to Rabs associated with the
same compartment or with compartments in dynamic
continuity. Examples are Rabaptins 4 and 5 (4,5), Rabenosyn 5 (6) and Rabip 40 (7), which are each able to interact
with both Rab4 and Rab5. These proteins are likely to be
involved in the formation and organization of Rab4 and Rab5
domains on endosomal membranes as well as in the coordination of endocytic and recycling trafficking pathways
(2,8). A protein, Rab coupling protein/Rab11-FIP1 (Family of
Interacting Protein 1), that binds to both Rab4 and Rab11
has also been identified (9), but its role in coupling Rab4 and
Rab11 function remains unclear (10). The second class
comprises proteins that behave as an effector for one Rab
and as an exchange factor (GTP exchange factor, GEF) for
another Rab. The identification of these proteins led to the
concept of a Rab cascade that explains how continuity can
be ensured along transport pathways (1). The best characterized is Sec2p that serves as an effector for Golgiassociated Ypt31p/Ypt32p and as a GEF for Sec4p present
on post-Golgi secretory vesicles (11). Another example is
the class C VPS–HOPS complex (Vacuole Protein SortingHomotypic fusion and vacuole protein sorting), a Rab7 GEF,
recently shown to be an effector of Rab5 and to participate
in the maturation of early Rab5-positive endosomes into late
Rab7-positive endosomes (12).
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Rab11 and Rab6 are predominantly associated with
membranes of the recycling endosomes (REs) and Golgi
complex, respectively (13–15). Numerous studies have
illustrated that members of the Rab11 subfamily,
Rab11A, Rab11B and the closely related Rab25, function
as regulators of endocytic membrane recycling in both
polarized and nonpolarized cells [for reviews, see Zerial
and McBride (2) and Hoekstra et al. (16)]. Work from our
laboratory has also shown that the overexpression of the
dominant-negative Rab11A mutant (S25N) or the addition of anti-Rab11 antibody to a permeabilized cell assay
inhibits the delivery of internalized B fragment of Shiga
toxin to trans Golgi network (TGN) membranes (17,18).
This suggests that Rab11A, in addition to its function in
recycling toward the plasma membrane, regulates transport events between REs and TGN. Rab6, for which three
isoforms (Rab6A, A0 and B) have been described, regulates intra-Golgi and Golgi to endoplasmic reticulum
retrograde transport (19–21). Rab6 also plays a role in
endosome to Golgi trafficking, as documented by its
participation in transport of Shiga and ricin toxins
(18,22). Depletion of Rab6A0 by small interfering RNA
(siRNA) or overexpression of the dominant-negative
Rab6A0 mutant (T27N) strongly inhibit the delivery of
Shiga toxin B fragment (STxB) to TGN (18,23). A likely
scenario is that TGN-associated Rab6A0 participates in the
tethering/docking of transport intermediates originating
from early endosomes and/or REs and carrying STxB.
This event requires the function of several effector
molecules that are recruited onto Golgi membranes by
Rab6:GTP, such as Rab6-interacting protein 2 (24), and
may serve as a link between Rab6A0 and the TGNlocalized t-SNARE complex, composed of Syntaxin 6,
Syntaxin 16 and Vti1a (18). Thus, Rab11 and Rab6 are
both involved in the retrograde transport of STxB. Furthermore, the finding that modulating the activities of
Rab6 and Rab11 using specific antibodies in the same
semi-in vitro system does not result in additive inhibition
of STxB transport suggests that these GTPases regulate
two sequential retrograde transport steps between REs
and TGN (18).
In addition to their role in membrane trafficking in interphasic cells, recent data indicate that Rab11 and Rab6 also
function in mitosis and cytokinesis. Rab11, in concert with
its effector Rab11-FIP3, is required for the delivery of
endocytic vesicles necessary for cell abscission at the
apex of the cleavage furrow (25). Rab6A, through its interaction with Rabkinesin-6/MKlp2 and Rab6A0 through its
interaction with GAPCenA and p150Glued, participates in
signaling pathways involved in the metaphase/anaphase
transition (unpublished results and 26).
The aim of this study was to identify the proteins that
could co-ordinate Rab6 and Rab11 function. Here, we
report the functional characterization of a protein, Rab6interacting protein 1 (R6IP1), originally identified as
a Rab6-binding protein in a yeast two-hybrid screen [clone
1386
B in (27)]. R6IP1, for which two isoforms are expressed
(R6IP1A and R6IP1B), also binds to Rab11A. We provide
evidence that R6IP1 acts in the Rab6 and Rab11 pathways both in interphase and during mitosis and
cytokinesis.
Results
Expression and structure of R6IP1
We first cloned R6IP1 complementary DNA (cDNA) from
a mouse brain library. Genome analyses revealed that
mouse R6IP1 gene is composed of 23 exons. Two splice
variants are found in the data banks, a short sequence
(accession number AJ245569), corresponding to splicing
of exon 2 that we named R6IP1A, and a longer sequence
(accession number BC060230), which includes all exons,
named R6IP1B. The 24 amino acid extension (exon 2)
present in R6IP1B is located at the N-terminus of the
protein (Figure 1A). In human Expressed Sequence Tag
(EST) databases, only the sequence of R6IP1B is present,
so we performed reverse transcriptase–polymerase chain
reaction (RT-PCR) using primers flanking the 24 amino acid
insertion in a variety of human cell lines to determine
whether R6IP1A is also expressed in humans. In the
human material tested, R6IP1B is by far the most abundant form of R6IP1. However, low amount of a RT-PCR
product corresponding to R6IP1A could be detected in
most cell lines, including HeLa cells (Figure 1B). The RTPCR experiments also revealed that in mouse, R6IP1A is
predominantly expressed in brain, whereas R6IP1B is the
major transcript in intestine (Figure 1B).
R6IP1 is found in most species, except in yeast Saccharomyces cerevisiae. Both isoforms are present in chick,
cow, zebra fish and tetraodon EST data banks. As in
humans, only the R6IP1B isoform has been identified in
rat and dog. On the contrary, insects such as bee,
anopheles and fly appear to only express R6IP1A.
Because of the divergence of the worm R6IP1 sequence
compared with the mammal proteins, it was difficult
to conclude which isoform is expressed in this organism.
Figure 1A shows the structure of the R6IP1A/B protein.
Its C-terminal half, which includes the Rab6-binding domain (Rab6BD) (see Figure 1), contains two RUN [RPIP8
(Rap2 interacting protein 8), UNC-14 and NESCA (new
molecule containing SH3 at the carboxy-terminus)] domains separated by a PLAT (Polycystin-1, Lipoxygenase,
Alpha Toxin) domain. RUN domains appear to be characteristic of proteins that interact with Ras-like GTPases
(28). In particular, they are present in Rabip4, a Rab4
effector and Connexin 43-interacting protein (CIP-85),
a Rab GTPase activating protein (GAP)-like protein (29,30).
PLAT domains are b-sandwich structures thought to
mediate interactions with lipids or membrane-associated
proteins (31). Finally, the N-terminal half of R6IP1 possesses
DENN (Differentially Expressed in Neoplastic versus Normal
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Functional Characterization of Rab6IP1
Figure 1: Structure and expression of R6IP1. A) Schematic representation of R6IP1A and R6IP1B isoforms and some of the truncated
forms used in this study. B) Reverse transcription–polymerase chain reaction (RT-PCR) analysis of R6IP1 expression in adult mouse tissues
and in human material. Mouse RT products of different tissues and of R6IP1A and R6IP1B plasmid cDNAs were amplified using mousespecific primers (left), and human RT products and mouse R6IP1A plasmid cDNA in the last lane on the right were amplified using humanspecific primers. IMR32 and HTB13 are neuroblastoma- and glioma-derived cell lines, Ewing E and F are Ewing tumors Erg and Fli, neuroblast
is neuroblastoma and De Luca is a rhabdoid tumor. Mr indicates molecular weight DNA markers, the size of which is indicated on the left.
Expected sizes of R6IP1A and R6IP1B RT-PCR products are 204 and 276 nt, respectively. aa, amino acid; Ct, C-terminus; Nt, N-terminus.
Cell) domains, which have been found in several proteins
involved in Rab-mediated processes or regulation of
mitogen-activated protein kinase signaling pathways (32).
However, it should be pointed out that R6IP1 does only bind
to Rab6-GTPgS in GST pull-down experiments performed at
higher stringency (>250 mM NaCl, data not shown).
R6IP1 interacts with both Rab6 and Rab11A
Yeast two-hybrid assay and biochemical pull-down experiments were performed to study the interaction of R6IP1A/B
with various Rab proteins. As shown in Table S1, the two
isoforms strongly interact in the yeast two-hybrid system
with the GTP-bound mutants of Rab6A, Rab6A0 and Rab6B
(Q72L) but not with dominant-negative Rab6A and Rab6A0
(T27N) mutants. A weak interaction was found with Rab6A
wild-type (wt) and a variable interaction with Rab6A0 wt.
This suggests that R6IP1 interacts with Rab6 in a GTPdependent manner. However, no nucleotide specificity was
found in glutathione S-transferase (GST) pull-down experiments performed in standard conditions. As shown in
Figure 2A, 2B (right panel), the same amount of R6IP1
present in mouse brain post-nuclear supernatant (PNS) was
bound to Rab6 incubated in the absence of nucleotide or
loaded with GDP or GTPgS. A similar result was obtained
using in vitro-translated R6IP1A or R6IP1B (data not shown).
No interaction was found between R6IP1 and Rab4, Rab5
or Rab7 (Table S1). In contrast, a specific interaction was
obtained with Rab11A by the yeast two-hybrid and GST
pull-down assays. However, in contrast to Rab6, Rab11A
binds to R6IP1A but not to R6IP1B (Table S1 and data not
shown). R6IP1A interacts with the two GTP-locked mutants of Rab11A, Rab11AQ70L and Rab11AS20V but not
with Rab11A wt and the dominant-negative mutant,
Rab11AS25N (Table S1). The nucleotide dependence of
Rab11A/R6IP1A interaction was preserved in GST pulldown experiments, as only Rab11A loaded with GTPgS is
able to bind R6IP1 present in mouse brain PNS (Figure
2A, 2B right panel) or to purified R6IP1A (data not shown).
We next compared the ability of Rab6 and Rab11 to bind
R6IP1A. As shown in Figure 2B (left panel), approximately
four times more R6IP1 can be bound to Rab6A/A0 :GTP
compared with Rab11A:GTP, suggesting that R6IP1 has
a better affinity for Rab6 than for Rab11A.
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Figure 2: R6IP1 interacts with Rab6 with no nucleotide specificity, whereas its binding to Rab11A is GTP dependent. Pull-down
experiments were performed using mouse brain PNS, mostly producing R6IP1A, as shown in Figure 1B. A) Glutathione S-transferase
(GST)–Rab fusion proteins were loaded with GDP or GTPgS or incubated in the absence of nucleotide (empty form). After incubation with
PNS in the presence or absence of nucleotide, R6IP1 bound to GST or GST–Rab was revealed by Western blot analysis using anti-R6IP1
antibody. Input represents a 5% load of the total PNS used in all conditions. B) Comparison of R6IP1 binding to Rab6 and Rab11. On the left
panel, R6IP1 bound to Rab loaded with GTPgS is shown, as either the ratio of Rab6A to Rab11 or that of Rab6A0 to Rab11. The means of
two experiments are shown. On the right panel, the ratio of R6IP1 bound to Rab:GTPgS to that bound to Rab:GDP is presented. For Rab6A,
the ratio is presented as means ! SEM of six experiments, for Rab6A0 nine experiments and for Rab11A eleven experiments. C) HeLa
cells were transfected with GFP–R6IP1A (lanes a–f) or with GFP–R6IP1B (lanes g–l) together with myc-Rab6AQ72L (lanes a and b, g and h)
or myc-Rab11AQ70L (lanes c and d, i and j) or myc-Rab11AS25N (lanes e and f, k and l). Untransfected cells were used as control (lanes m
and n). Cells lysates were immunoprecipitated using a goat anti-myc antibody. GFP fusion proteins were detected using an anti-GFP
antibody on the upper half of the blot; immunoprecipitation efficiency was controlled using goat anti-myc on the lower half of the gel.
To, 1/30 of the cell lysate before immunoprecipitation; IP, immunoprecipitate; WB, western blotting.
To further establish the interaction between R6IP1 and
either Rab6 or Rab11A, we performed a series of coimmunoprecipitation experiments in cells coexpressing
myc-Rab6A or myc-Rab11A constructs and either green
fluorescent protein (GFP)–R6IP1B or GFP–R6IP1A. GFP–
R6IP1A (Figure 2C, lanes a and b) and GFP–R6IP1B
(Figure 2C, lanes g and h) could be coimmunoprecipitated
with myc-Rab6AQ72L and myc-Rab6A‘Q72L (data not
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shown). GFP–R6IP1A was also coimmunoprecipitated
with myc-Rab11AQ70L but not with myc-Rab11AS25N,
as expected (Figure 2C, lanes c and d, e and f). Lower but
detectable amounts of GFP–R6IP1B were found in complexes with myc-Rab11AQ70L (Figure 2C, lanes i and j). In
contrast, no GFP–R6IP1B was immunoprecipitated with
myc-Rab11AS25N (Figure 2C, lanes k and l). Although no
interaction was detected in vitro between Rab11A and
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Functional Characterization of Rab6IP1
Figure 3: The recruitment of R6IP1 to
Golgi membranes is Rab6-dependent. A) HeLa cells were transfected
with GFP–R6IP1A. Endogenous Rab6
was revealed with a specific antibody.
B, C) HeLa cells were treated for 2 days
with Rab6A/A0 siRNA and then transfected with GFP–R6IP1A alone (B) or
with GFP–R6IP1A and wild-type (wt)
Rab6A (C). Endogenous (B) and overexpressed (C) Rab6 was revealed with
a specific antibody. D, E) HeLa cells
were cotransfected with wt CFP–Rab6A
and either YFP–R6IP1A (panel D) or
YFP–R6IP1B (panel E). Each image represents a single confocal section (A–C)
or a projection of confocal sections (D,
E). Scale bars, 10 mm.
R6IP1B, these experiments suggest that both proteins can
directly or indirectly interact in vivo. Higher amounts of
GFP–R6IP1A were found associated with Rab6AQ72L
than with Rab11AQ70L, in good agreement with the
results obtained in GST pull-down assays (Figure 2B, left
panel).
We next constructed a series of truncation mutants to map
the Rab-binding domain(s) of R6IP1 (Figure 1A and data
not shown). The minimal Rab6BD was identified using the
yeast two-hybrid assay (Table S1) and is constituted by the
segment encompassing residues 683–1066 (R6IP1A) or
707–1090 (R6IP1B). This domain includes a RUN–PLAT
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domain (Figure 1A), present in both R6IP1A and R6IP1B.
Any further truncation of the Rab6BD led to a loss of Rab6/
R6IP1 interaction. Strikingly, an interaction between
R6IP1A and Rab11A could only be obtained with the entire
protein (Table S1). This suggests that secondary structures
of R6IP1A, including both N and C extremities, are required
for Rab11A recognition.
R6IP1 is recruited by Rab6 on Golgi membranes
and by Rab11 on REs
Endogenous R6IP1 was visualized by immunofluorescence on HeLa cells using a polyclonal rabbit antibody
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raised against recombinant full-length R6IP1A. This antibody also recognizes R6IP1B (data not shown). A faint and
diffuse cytosolic staining was observed (data not shown).
Fractionation experiments confirmed that endogenous
R6IP1 is mainly found in the cytosol (Figure S1). The
cytoplasmic localization of R6IP1 is consistent with the
fact that R6IP1 does not bear any putative transmembrane
domain. However, by moderately overexpressing GFP–
R6IP1A or GFP–R6IP1B fusion proteins, part of GFP–
R6IP1 was found to colocalize with Rab6 at the Golgi
complex (Figure 3A). As analyzed by immunogold labeling
on ultrathin cryosections, the majority of GFP–R6IP1A and
GFP–R6IP1B revealed by anti-GFP antibodies were detected on Golgi stacks and on closely apposed tubulovesicular elements positive for the TGN marker TGN46 (see
Figures 6A–C and S3, for quantification).
In addition, the expression at moderate levels of Rab6A/A’
wt or Q72L mutants (data not shown) together with
R6IP1A/B resulted in a nearly complete colocalization of
both proteins at the Golgi complex, as illustrated in
Figure 3D,E for cyan fluorescent protein (CFP)–Rab6A wt
and yellow fluorescent protein (YFP)–R6IP1A or YFP–
R6IP1B. In contrast, overexpressed R6IP1A/B remained
cytosolic in cells cotransfected with Rab6A/A0 T27N mutants (data not shown).
To investigate whether Rab6 is required to localize
R6IP1A/B to the Golgi complex, endogenous Rab6A
and Rab6A0 were depleted by siRNAs. In Rab6-depleted
cells, overexpressed GFP–R6IP1A was found to be
strictly cytosolic and no concentration was observed in
the Golgi region (Figure 3B). In contrast, the re-expression of Rab6A wt (Figure 3C) or Rab6A0 wt (data not
shown) in Rab6A/A0 -depleted cells restored the localization of GFP–R6IP1A to the Golgi (Figure 3C). Similar
results were found for GFP–R6IP1B (data not shown).
Finally, cells depleted of Rab11A by siRNAs showed no
alteration in the Golgi localization of R6IP1A (Figure 4A).
Altogether, the above experiments indicate that R6IP1A/
B is recruited to the Golgi in a Rab6-dependent manner.
In addition, the recruitment of R6IP1A/B to the Golgi
does not depend on its interaction with endogenous
Rab11A.
Figure 4: R6IP1 binds to Rab11-positive compartments in cells overexpressing Rab11A. A, B) HeLa cells
were treated for 2 days with Rab11A
siRNA and then transfected with GFP–
R6IP1A alone (panel A), or with GFP–
R6IP1A and wild-type (wt) Rab11A (panel
B). Endogenous (A) and overexpressed
(B) Rab11 was revealed with a specific
antibody. C, D) HeLa cells were cotransfected with wt CFP–Rab11A and YFP–
R6IP1A (panel C) or wt CFP–Rab11A and
YFP–R6IP1B (panel D). Each image is a
single confocal image (A) or a projection
of confocal sections (B, C, D). Arrowheads in insets point to peripheral structures costained with Rab11 and R6IP1.
Scale bars, 10 mm.
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Functional Characterization of Rab6IP1
Figure 5: R6IP1 overexpression alters the distribution of peripheral
REs. GFP–R6IP1A (panels A, C and E)
or GFP–R6IP1B (panels B and D) constructs were overexpressed for 24 h.
Cells were then processed for immunofluorescence using specific antibodies against Rab11 (panels A and B), TfR
(panels C and D) or EEA1 (panel E).
Images are single confocal sections.
Scale bars, 10 mm.
A strikingly different picture was obtained in cells coexpressing Rab11A wt or Rab11AQ70L with R6IP1A/B. In
these cells, a strong overlap was observed between
R6IP1A/B and Rab11 staining. This is illustrated in Figure 4B,
showing a cell cotransfected with Rab11A wt and GFP–
R6IP1A after Rab11-depletion by siRNA. GFP–R6IP1A was
found to colocalize with Rab11A both in the pericentriolar
region of the cells and in more peripheral structures
characteristic of the recycling compartment in HeLa cells
(Figure 4B, arrowheads in insets). Thus, higher amounts of
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Rab11A within the cells favor the recruitment of R6IP1A to
Rab11-positive compartments. To further confirm this
observation, we coexpressed YFP–R6IP1A with CFP–
Rab11A wt. As illustrated in Figure 4C, a nearly complete
colocalization was observed between the two proteins,
both in pericentriolar and in peripheral REs (arrowheads in
insets). As the Golgi complex and the Rab11-positive
pericentriolar recycling compartment are located in the
same area of the cell (illustrated in Figure S2 for a cell
expressing YFP–R6IP1A and CFP–Rab11A), it was difficult,
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Figure 6: R6IP1 is mainly localized
to the Golgi apparatus. Ultrathin cryosections of HeLa cells expressing
GFP–R6IP1A (A, C and D) and GFP–
R6IP1B (B) were single immunogold
labeled for GFP (A and B) or double
immunogold labeled for GFP and
TGN46 (C) or GFP and HRP (D). Antibodies were visualized with protein A
conjugated to 15- or 10-nm gold particles as indicated. In D, cells were
allowed to internalize Tf coupled to
HRP for 45 min before fixation. A, B)
Green fluorescent protein–R6IP1A and
GFP–R6IP1B are detected not only in
the cisternae of the Golgi apparatus
(GA) but also in closely apposed vesicular elements (arrows in B). C) As in A,
note the localization of GFP–R6IP1A in
the GA, including in vesicles at the
trans side of the GA (arrows). In addition, GFP–R6IP1A is also present in
vacuolar endosomal structures displaying intraluminal membranes (arrowheads). D) Internalized Tf–HRP can be
detected close to the GA (arrowheads), but it is not present in the GA
cisternae, containing the bulk of GFP–
R6IP1A. PM, plasma membrane. Scale
bars, 200 nm.
however, to determine whether R6IP1A was still associated
with Golgi in cells overexpressing active Rab11. Similar
results were obtained after coexpression of Rab11A and
R6IP1B (Figure 4D, cell on the left). Nevertheless, the
colocalization between YFP–R6IP1B and CFP–Rab11A appeared less complete than the one observed between YFP–
R6IP1A and CFP–Rab11A (Figure 4D, cell on the right).
Altogether, the above results suggest that R6IP1 behaves
as a Rab6-interacting protein in vivo and is targeted to the
Golgi in a Rab6-dependent manner. Moreover, R6IP1 can
associate to Rab11-positive compartments when levels of
Rab11A are increased within the cells.
The overexpression of R6IP1 affects the spatial
distribution of the REs
We noticed that the overexpression of R6IP1 alters endogenous Rab11 staining. As shown in Figure 5A,B (outlined
cells), R6IP1 overexpression resulted in a depletion of
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peripheral Rab11-positive compartments, including those
present in cell tips, and in a concomitant concentration in
the pericentriolar area. Transfected cells also displayed an
accumulation of transferrin receptor (TfR) in the pericentriolar area (Figure 5C,D). In contrast, the distribution of the
early endosome marker Early Endosomal Antigen 1 (EEA1)
(Figure 5E) as well as the Golgi markers CTR433 and
GM130 (data not shown) remained unaffected.
At the ultracryomicroscopy level, endosomal compartments with a vacuolar appearance and with very few
internal membranes were found close to the Golgi in
cells overexpressing R6IP1 (illustrated in Figure 6C,D
for R6IP1A). R6IP1A molecules were present on the
limiting membrane of these compartments that often
contained internalized Horse Radish Peroxydase (HRP)–
transferrin (Tf) (Figure 6D). However, labeled membranes
with R6IP1A/B and HRP–Tf appeared distinct from
Golgi cisternae, and no HRP labeling could be found
within them.
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Functional Characterization of Rab6IP1
Thus, the overexpression of R6IP1A/B leads to concentration of Rab11- and TfR-positive recycling compartments to the pericentriolar area but does not seem to
induce fusion events between endosomal and Golgi
membranes.
In vivo interactions between Rab6 and Rab11A in
the presence of R6IP1 by two-photon fluorescence
resonance energy transfer/fluorescence lifetime
imaging
The above results suggest that the overexpression of
R6IP1A/B brings closer Rab6- and Rab11-positive compartments. To investigate whether an interaction between Rab6
and Rab11A after R6IP1A/B overexpression could mechanistically explain this topological gathering, we set up twophoton fluorescence resonance energy transfer (FRET)/
fluorescence lifetime imaging (FLIM) experiments in living
cells. A first series of FLIM experiments was performed in
cells expressing only CFP–Rab6A to obtain a reference
value for the mean fluorescence lifetime of CFP without
any FRET (Figure 7A, top left, and Figure 7B, first row). An
average value of 2.4 nanoseconds was obtained, which
corresponds to the expected and published value for the
CFP (33). We next measured the fluorescence lifetime of
CFP in cells coexpressing CFP–Rab6A or CFP-Rab6A0 and
YFP–Rab11A. Image analysis was performed in the pericentriolar region of the cells, where the major part of Rab6
and Rab11A signal is detected (Figure 7A, top right).
However, the low signal intensity and the acquisition time
of FLIM images (300 seconds) precluded a reliable analysis
of the fast-moving peripheral Rab11-positive vesicles. CFP
lifetime displayed a value similar to the reference value for
CFP alone (Figure 7B, first row, and Figure 7C), indicating
that no interaction between Rab6A/A0 and Rab11A in transfected cells could be detected.
In cells cotransfected with CFP–Rab6A or CFP–Rab6A0
and YFP–R6IP1A, values lower than 2.4 nanoseconds
(Figure 7B, second row) were obtained, indicating that
FRET occurred between the two fluorophores CFP and
YFP and thus that Rab6A/A0 and R6IP1A interact. The
broad distribution of the values shown on histograms may
be due to variations in the relative position of the proteins
and fluorophores. As the FRET efficiency varies as the
inverse 6th power of the distance between the donor and
the acceptor, slight changes can lead to significant
variations in fluorescence lifetime (34). Similar experiments
were performed in CFP–Rab11A-expressing cells cotransfected with YFP–R6IP1A (Figure 7A, bottom left). As shown
in Figure 7B (second row), values lower than 2.4 nanoseconds were also found. Thus, the above results confirm the ability of Rab6A/A0 and Rab11A to interact in vivo
with R6IP1A.
We then measured the lifetime in cells overexpressing
R6IP1A together with CFP–Rab6 and YFP–Rab11A. The
CFP lifetime value was reduced in about 40% of cells,
indicating that CFP–Rab6 and YFP–Rab11A were close
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enough for FRET to occur (Figure 7A, bottom right, Figure
7B, third row and Figure 7C). Similar results were obtained
in cells expressing YFP–Rab11AQ70L instead of YFP–
Rab11A wt (Figure 7C). In contrast, no FRET was observed
in cells expressing YFP–Rab11AS25N (Figure 7B,C), in good
agreement with previous data showing the GTP nucleotide
specificity of the Rab11A/R6IP1A interaction (Figure 2).
As a control for specificity, we performed experiments
with cells expressing YFP–Rab4A, a Rab protein that
partially localizes to the same compartments as Rab11A
(8). No FRET occurred between YFP–Rab4A and CFP–
Rab6A when R6IP1A was overexpressed (Figure 7C),
supporting the specificity of the ternary association
between Rab6, Rab11A and R6IP1.
The above results thus indicate that R6IP1A can specifically trigger in vivo the formation of complexes containing
Rab6 and Rab11A.
R6IP1 may oligomerize to bind both Rab6 and Rab11A
There are several possibilities to explain how R6IP1 can
promote an interaction between Rab6 and Rab11A. One is
that a single R6IP1 molecule is able to bind simultaneously
to Rab6 and Rab11A. To investigate this, GST pull-down
assays were performed by adding R6IP1A/B from various
sources and Rab11A loaded with GTPgS sequentially to
GST-Rab6 or by adding simultaneously the two proteins.
However, no concomitant binding of R6IP1A/B and
Rab11A was obtained (data not shown). Another possibility is that R6IP1 has to oligomerize in order to bind both
Rab6 and Rab11A. To test this hypothesis, we performed
a series of coimmunoprecipitation experiments using
various R6IP1A/B constructs. As shown in Figure 8 (lanes
a and b, c and d), full-length R6IP1A and R6IP1B can be
coimmunoprecipitated with their respective N-terminal
halves (Nt, Figure 1A). The N-terminal regions of R6IP1
can also interact in vivo with each other, as illustrated by
coimmunoprecipitation of myc-tagged R6IP1A-Nt with
GFP-tagged R6IP1A-Nt in cells expressing both constructs
(Figure 8, lanes e and f). Under the same conditions, the
C-terminal regions (Ct, Figure 1A) did not show any interaction (data not shown). This suggests that R6IP1 molecules can form in vivo homo-oligomers via their Nt domain.
R6IP1A and R6IP1B also have the capability to form
hetero-oligomers. The two proteins were found to weakly
interact by yeast two-hybrid assay (Table S1), and GFP–
R6IP1B can be coimmunoprecipitated with myc-tagged
R6IP1A from cell lysates expressing both constructs
(Figure 8, lanes g and h). Finally, we tested whether
intramolecular interactions within R6IP1 take place. Weak
but significant interactions over background were found
between Ct and Nt halves of R6IP1A or B (Table S1). This
interaction was confirmed by coimmunoprecipitation experiments (Figure 8, lanes i and j).
These data suggest that R6IP1 form in vivo homo- or
hetero-oligomers that allow binding of Rab6 and Rab11A
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Figure 7: Legend on next page.
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Functional Characterization of Rab6IP1
Figure 8: R6IP1 can form in vivo
homo- or hetero-oligomers. HeLa
cells were cotransfected with GFP–
R6IP1B (g and h) or GFP–R6IP1B Nt
(c and d) or GFP–R6IP1A (a and b) or
GFP–R6IP1A Nt (e and f) or GFPR6IP1Ct (i and j), together with mycR6IP1B Ct (c and d) or myc-R6IP1A (g
and h) or with myc-R6IP1A Nt (a and b,
e and f, i and j). Cell lysates were then
immunoprecipitated with a goat antimyc antibody, and GFP fusion proteins
were detected by western blotting
(WB) using an anti-GFP antibody.
Molecular weight markers are indicated
on the left (in kDa), the position of
proteins of interest noted on the right.
Nt, N-terminal; Ct, C-terminal; To, 1/30
of the cell lysate before immunoprecipitation; IP, immunoprecipitate.
within the same complex. They also provide an explanation
about the observation that GFP–R6IP1B, which does not
interact in vitro with Rab11A, still associates to Rab11positive compartments in cells overexpressing Rab11A wt
or Rab11AQ70L (Figure 4). R6IP1B may indeed form
a complex with R6IP1A, which allows its binding to
Rab11A. Finally, the existence of interactions between
the Nt and the Ct regions suggests that R6IP1 may adopt
a folded conformation.
Overexpression of R6IP1 increases the rate of
Rab11-dependent recycling of Tf
We then investigated whether the redistribution of peripheral Rab11-positive compartments toward the pericentriolar area affects transport events associated with REs. We
first monitored the transport of STxB between endosomes
and Golgi, shown to involve in part Rab11-positive compartments (17). No significant changes in the kinetics of
delivery of internalized Cy3-STxB to the TGN was observed
after overexpression of R6IP1A/B (data not shown). The
kinetics of the appearance of ts-O45 vesicular stomatitis
virus G protein to the cell surface, that transits in part
through the REs on its way to the plasma membrane (35),
also remained unaffected (data not shown).
However, an effect was observed on the rate of Tf
recycling. After moderate overexpression of GFP–R6IP1A
or GFP–R6IP1B, Tf uptake was comparable to that in
control cells, but after a chase time of 30 min, Tf recycling
was found to be slightly but consistently higher (10%)
(Figure 9; Table S2). The colocalization of internalized Tf
and Rab11 has been shown to be maximal after 30 min to
1 h in various cell types (14,36). In addition, an effect of
Rab11 mutants on Tf recycling can be clearly detected only
after 30-min chase period following Tf uptake (14,17). This
suggests that the overexpression of R6IP1A/B affects the
Rab11-dependent step of Tf recycling. It has been documented that about 20% of Tf recycles through the Rab11
pathway in HeLa cells (17) and other cell types (37). Thus,
this indicates that the overexpression of R6IP1 increases
by about 50% the rate of recycling of internalized Tf that
passes through the Rab11-positive endosomes.
The above results suggest that the concentration of
Rab11-positive compartments in the pericentriolar area
on R6IP1 overexpression increases the efficiency of
TfR recycling but does not affect the transport pathway between endosomes and Golgi/TGN monitored by
Shiga toxin.
R6IP1 function is required for mitotic progression
Interestingly, a strong effect of R6IP1 overexpression was
observed during mitosis and cytokinesis. For these experiments, cells were transfected with R6IP1A/B and analyzed
by time-lapse phase-contrast videomicroscopy. As shown
in Table 1, a significant proportion of cells were blocked in
Figure 7: Interaction between Rab6, R6IP1 and Rab11A in living cells by FRET/FLIM. A) Intensity (left) and FLIM images (middle) of
a HeLa cell expressing CFP–Rab6A (top left), CFP–Rab11A/YFP–R6IP1A (bottom left), CFP–Rab6A/YFP–Rab11A (top right) or CFP–Rab6A/
R6IP1A/YFP–Rab11A (bottom right). The color coding of the FLIM image indicates the fluorescence lifetime value following the total
distribution of lifetime in the image between 0 and 4000 picoseconds (right). B) Distributions of lifetime values over n cells expressing from
left to right and from top to bottom CFP–Rab6A alone, CFP–Rab6A/YFP–Rab11A, CFP–Rab6A0 /YFP–Rab11A, CFP–Rab6A/YFP–R6IP1A,
CFP–Rab6A0 /YFP–R6IP1A, CFP–Rab11A/YFP–R6IP1A or CFP–Rab6A/R6IP1A/YFP–Rab11A, CFP–Rab6A0 /R6IP1A/YFP–Rab11A and CFP–
Rab6A/R6IP1A/YFP–Rab11AS25N. C) Graphs represent the synthesis of the mean filtered values of fluorescence lifetimes (left) and
estimated FRET efficiency (right) ! SD obtained for the combinations of protein overexpression shown in B. Graphs also included data
obtained for cells expressing CFP–Rab6A/R6IP1A/YFP–Rab11Q70L (n ¼ 20), CFP–Rab6A/R6IP1A/YFP–Rab4A (n ¼ 16) and CFP–Rab6A0 /
R6IP1A/YFP–Rab4A (n ¼ 11).
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Figure 9: The overexpression of R6IP1 affects Tf recycling. HeLa cells transfected with GFP–R6IP1A or GFP–R6IP1B were loaded with
Alexa633–Tf, as described in Material and Methods and chased for the various times indicated on the figure. Cells producing either GFP–
R6IP1A (left) or GFP–R6IP1B (right) were sorted as no GFP–R6IP1 expression (control, blue curves) or as moderately expressing cells
(GFP–R6IP1A or GFP–R6IP1B, red curves). Results were expressed as the percentage of Alexa633–Tf associated to cells at each timepoint compared with the maximum Alexa633–Tf associated to cells at t ¼ 0. Values are expressed as means ! SEM of four experiments
for GFP–R6IP1A-expressing cells and of five experiments for GFP–R6IP1B-expressing cells. Paired Student’s t-tests are indicated in Table S2.
metaphase 24–48 h after transfection. This phenotype
was reminiscent to that observed after depletion of Rab6A0
or overexpression of the dominant negative form of
Rab6A0 (26). In addition, the percentage of binucleated
cells was increased after 3 days, suggesting a defect in
cytokinesis (Table 1).
To further understand these phenotypes, endogenous
R6IP1 was depleted by siRNAs. As estimated by Western
blotting, the transfection of cells with two different siRNAs
(siR6IP1-1 and siR6IP1-2, see Material and Methods) led to
an efficient knockdown of R6IP1 expression (Figure 10A).
No major effects on the morphology of the Golgi complex
or of endocytic compartments using various markers was
Table 1: Effects of the overexpression of GFP–R6IP1A and GFP–
R6IP1B on cell cycle. Cells were transfected with a control
plasmid, GFP–R6IP1A or GFP–R6IP1B and imaged by time-lapse
phase-contrast videomicroscopy. The percentage of cells arrested
in metaphase was measured by analyzing the recorded movies. In
parallel, cells were fixed 3 days after transfection and multinucleated cells were counted. Results are representative of two to
three independent experiments. The number of cells analyzed
ranges from 59 to 224. Results are expressed as means ! SEM
Control plasmid
GFP–R6IP1A
GFP–R6IP1B
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% Of cells
arrested in mitosis
% Of multinucleated
cells
3.9 ! 0.1
25 ! 5
13 ! 0.1
2.3 ! 0.3
14.2 ! 1.7
9.2 ! 1.2
observed in interphasic cells present in the R6IP1-depleted
cell population (data not shown). Transport of internalized
STxB toward the Golgi and Tf uptake and recycling did not
also seem to be significantly affected. However, time-lapse
videomicroscopy experiments showed that about 50% of
R6IP1-depleted cells with siR6IP1-1 and siR6IP1-2 were
arrested in metaphase and were unable to exit mitosis
compared with 16% in control cells (Figure 10B, a). Arrested
cells, which exhibited a normal alignment of their chromosomes at the metaphase plate (Movie S3), died after a few
hours (Figure 10B, b and Movie S2 and S3). We analyzed
this phenotype in further detail using the siRNA approach.
Cells arrested in metaphase displayed a normal mitotic
spindle, as judged by b-tubulin staining (data not shown).
The majority of the cells (67%) showed three or more
kinetochores labeled with the Mad2 antibody, suggesting an
activation of the Mad2 spindle checkpoint (Figure 10C). This
was further confirmed by silencing Mad2 by siRNA, which
resulted in a 50% decrease of the metaphase arrest induced
by the treatment with siR6IP1-1 (Figure 10D). Thus, the
mitotic arrest observed in R6IP1-depleted cells is very
similar to that obtained after alteration of Rab6A0 function,
shown to induce a Mad2-dependent metaphase arrest (26).
In addition, R6IP1-depleted cells displayed cytokinesis defects. We observed cells that start to divide normally but
undergo re-fusion after an ineffective cleavage, giving rise
to a binucleated cell (Figure 10E). Following a 72 h depletion with both R6IP1 siRNAs, 5% of cells were binucleated
(Figure 10F). Interestingly, Wilson et al. have reported that
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Functional Characterization of Rab6IP1
Figure 10: Legend on next page.
Rab11 depletion or overexpression of Rab11 S25N in HeLa
cells also results in cytokinesis defects, although the
percentage of binucleated cells was significantly higher in
their experiments (25).
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Figure S4 illustrates the localization of GFP–R6IP1A in
mitotic cells. Up to anaphase, R6IP1A displays a diffuse
cytosolic localization. Such a localization is also found for
Rab6 (26; Figure S4) and Rab11, even though some
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Miserey-Lenkei et al.
concentration can be detected around the spindle poles, as
previously reported (38; Figure S4). Interestingly, in telophase and cytokinesis, GFP–R6IP1A associates to reforming Golgi and concentrates at the intracellular bridge where
it is found colocalized with Rab6- and Rab11-positive
structures.
although the overall identity between the two proteins is
only 5.2%. However, we have not been able to detect any
GEF activity in vitro on Rab6 (data not shown). It should also
be pointed out that the exact domain performing GEF
function in Rab3 GEF has not been precisely mapped and
that DENN domains are also not found in other Rab GEFs.
Altogether, our data suggest that low amounts of R6IP1
are sufficient for its function in interphase and that highest
levels of depletion give rise to strong mitotic phenotypes.
They show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which the role
of Rab6 and Rab11 has been previously documented.
Without ruling out the above hypothesis, it is more likely
that R6IP1 acts as a Rab6 effector recruited by Rab6 on
Golgi membranes. This is consistent with the findings that
R6IP1 is a cytosolic protein and that Rab6 depletion inhibits
targeting of overexpressed R6IP1 to Golgi membranes.
Along with this hypothesis, it would be the Rab6–R6IP1
complexes that interact, once formed, with Rab11. We
obtained no evidence that a single R6IP1 molecule can
bind simultaneously to Rab6 and Rab11, suggesting that
Rab6/Rab11 interaction occurs through homo- and/or
hetero-oligomerization of R6IP1 molecules. Rab6 binds to
the C-terminal part of R6IP1, which has the potential to
interact with its own N-terminus. It is thus possible that
Rab6 binding leads to the opening of one R6IP1 molecule,
which allows the binding of another R6IP1A or R6IP1B
molecule and then of Rab11. R6IP1A and B are expressed
at different levels depending on cell types and organisms.
In addition, differences exist in the ability of Rab11A to
bind either R6IP1A or R6IP1B in vitro, indicating that the
insertion of a 24 amino acid stretch in R6IP1B affects the
R6IP1/Rab11A interaction. These results suggest that
Rab6/Rab11A interaction could be subjected to additional
levels of regulation through the formation of R6IP1 homoor hetero-oligomers of different composition. Further experiments will be required to clarify these hypotheses.
Discussion
The relationships that exist between the Golgi complex and
the REs are still poorly understood. However, it is now clear
that the connection between these compartments occurs
both in the retrograde and in the anterograde directions. REs
may serve as a transit station toward the TGN for internalized toxins such as Shiga toxin and for resident TGN proteins
such as TGN38 recycled back from the plasma membrane
(39,40). Recent studies have highlighted that REs may also
serve as a hub for the sorting of newly synthesized and
endocytosed proteins (35). The identification of two proteins, R6IP1A and R6IP1B, that interact with both Rab6 and
Rab11 provides to our knowledge the first molecular link
between the two compartments.
Several hypotheses are drawn to explain how R6IP1 may
function in concert with Rab11 and Rab6. Taking into
account the experimental evidence that Rab11A and Rab6A0
regulate two sequential transport steps of STxB fragment
between endosomes and Golgi (18), R6IP1 could function in
a Rab cascade mechanism between these two compartments. R6IP1 could be recruited on REs by Rab11A:GTP
and then acts as an exchange factor for Rab6. It is
noteworthy that the N-terminal half of R6IP1 shows similarity through its DENN domains with Rab3 GEF (41),
In addition to REs, Rab11 was found to be associated with
Golgi and/or TGN membranes in some cell types (14,42).
However, endogenous Rab11 is not detected at the
immunofluorescence level on Golgi/TGN membranes in
HeLa cells (17). In these cells, the Rab11-positive compartment appears more scattered in the cytoplasm than in
other cell types, such as Chinese Hamster Ovary (CHO) or
PC12 cells, where it is predominantly concentrated in the
Figure 10: R6IP1 is required for metaphase/anaphase transition and completion of cytokinesis. A) 72 h after transfection with siRNA
that target R6IP1 (namely R6IP1-1 or -2) or control siRNA, cell lysates were subjected to Western blotting analysis and probed with an antiR6IP1 antibody (top) or anti-Rab11 antibody (bottom). B) a: Percentage of cells arrested in metaphase after transfection of control and R6IP1-1
or -2 siRNA. b: HeLa cells were transfected with control (top) or R6IP1-1 siRNA (bottom) and imaged for 72 h using time-lapse phase-contrast
videomicroscopy. Arrowheads point to R6IP1-depleted cells arrested in metaphase. Note the alignment of chromosomes on the metaphase
plate (Movie S3). The numbers correspond to the time in hours after the beginning of the recording. The corresponding movies (Movies S1,S2
and S3) are presented in supplementary online material. C) Maximal intensity projection through the z-axis of deconvolved images stacks of
metaphasic cells 48 h after transfection with R6IP1 siRNA. Cells were costained with a CREST (calcinosis, Raynaud phenomenon, esophageal
dismotility, sclerodactyly, telangiectasia) serum (left, green), anti-Mad2 antibody (middle, red) and DAPI (40 ,6-diamidino-2-phenylindole) (right,
blue). Higher magnification for details of localization of CREST and Mad2 staining is shown in insets. Bar, 10 mm. D) Percentage of cells
arrested in metaphase after cotransfection of R6IP1-1 siRNA with either control or Mad2 siRNA. Results are expressed as a ratio to the
percentage found in cells cotransfected with R6IP1 and control siRNA. E) Details of a cell undergoing cytokinesis defect after R6IP1-1 siRNA.
The numbers correspond to the time in hours after the beginning of the recording. Arrowheads point to a cell undergoing a cytokinesis defect.
The corresponding movie (Movie S4) is presented in supplementary online material. F) Percentage of multi-nucleated cells (counted on fixed
sample) after transfection of control or R6IP1-1 and -2 siRNA. In B, D and F, number of cells analyzed ranged from 48 to 86. Results are
representative of two to three independent experiments and are presented as means ! SEM.
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Functional Characterization of Rab6IP1
pericentriolar region. We observed that the overexpression
of R6IP1 results in a depletion of peripheral Rab11-positive
REs, indicating that increased amount of R6IP1 affects
their dynamics. A likely explanation of this effect is that
Rab6–R6IP1 complexes interact with Rab11 when Rab11positive peripheral structures move from the periphery to
the cell center. In this case, excess amount of R6IP1 would
be expected to shift the steady-state repartition of peripheral and pericentriolar REs. Thus, a tempting hypothesis is
that Rab6–R6IP1 complexes participate in the tethering/
docking of transport intermediates that carry STxB
between endosomes and Golgi/TGN membranes. This
hypothesis is strongly supported by the results of FLIM–
FRET analysis performed on living cells. It would also fit
with previous results, indicating that part of STxB en route
to TGN transits through REs (18). We do not exclude,
however, other mechanisms accounting for the redistribution of peripheral REs. This phenotype is reminiscent of
that obtained by overexpressing GTPase-deficient mutant
of Golgi-associated Rab34, shown to mediate a long-range
regulation of the spatial distribution of lysosomes (43).
Rab34 was proposed to recruit the Rab7 effector RabInteracting Lysosomal Protein (RILP), which results in the
modulation of the activity of the dynein–dynactin complex
to promote the migration of lysosomes toward the minus
end of microtubules (43). As Rab6 has been shown to
interact with the same motor protein complex (26,44,45),
a similar scenario could be envisioned for the effect of
R6IP1 on the Rab11-positive compartment distribution.
The concentration of Rab11-positive recycling compartments in the pericentriolar area on R6IP1 was correlated to
a significant acceleration of TfR recycling. One hypothesis
is that this effect is because of a shunting of the TfR into
the recycling pathway that does not require passage
through REs, i.e. early endosomes. However, the effect
of R6IP1 overexpression was detected after a 30-min
chase period, which is not compatible with the fast
Rab4-dependent recycling step of internalized Tf. Another
possibility is that TfR recycles via an alternative pathway.
For instance, TfR could recycle faster because it enters the
secretory pathway at the Golgi/TGN level. However, we
obtained no evidence at the ultracryomicroscopy level that
R6IP1 overexpression induces a fusion between REs and
Golgi/TGN membranes. A concentration of the REs in the
perinuclear region may also favor the association of transport intermediates carrying TfR toward the cell surface
with microtubules. Finally, it should be pointed out that the
concentration of Rab11 and TfR in the pericentriolar
area was documented after the overexpression of arfophilin-2 and a tail chimera of myosin Vb, two other Rab11
effectors (46–48). Whereas the recycling of TfR is not
significantly perturbed by the overexpression of arfophilin2, it is retarded in cells expressing myosin Vb mutant. A
better understanding of the molecular mechanisms involved
in slow TfR recycling should help us to understand the
various effects induced by the three Rab11 effectors. In
particular, it will be of interest to investigate the relationship
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between R6IP1 and myosin Vb, shown to function in traffic
between peripheral and pericentriolar REs (48).
Our results indicate that R6IP1 also plays a role in the Rab6
and Rab11 pathways during mitosis and cytokinesis. The
block in metaphase induced by R6IP1 depletion is very
similar to the one obtained after depletion of Rab6A0 ,
leading to an activation of the Mad2 spindle checkpoint
(26). This suggests that R6IP1 acts as a Rab6A0 effector at
the metaphase/anaphase transition. The observation that
R6IP1 overexpression has the same effect as its depletion
could be explained by an inhibitory effect on the interaction
between Rab6A0 and another effector required for Rab6A0
function. We have shown that p150Glued, a subunit of the
dynein–dynactin complex, acts in the Rab6A0 pathway and
that Rab6A0 /p150Glued interaction is important for the
dynamics of the dynein–dynactin complex at the kinetochores (26). Rab6A0 pathway also involves GAPCenA (49),
acting in this case not as a GAP but rather as an effector
protein. We have proposed that GAPCenA interacts
through its N-terminal phosphotyrosine-binding domain
with other regulatory proteins involved in Rab6A0 function
(26). R6IP1 could be one of these proteins. R6IP1 is
present in the interphasic cytosol in fractions of very high
molecular mass containing GAPCenA and p150Glued (data
not shown). Future studies should establish whether it is
also the case in mitosis.
A significant proportion of R6IP1-depleted cells or R6IP1overexpressing cells, likely those that succeeded to pass the
metaphase block, were binucleated. This suggests that
R6IP1, as previously documented for Rab11 (25), is required
for cytokinesis. The exact role of Rab11 in cytokinesis is not
yet fully understood. It has been proposed that Rab11–FIP3
complexes are involved in the tethering/fusion of vesicles at
the apex of the cleavage furrow, allowing the delivery of
proteins/membranes necessary for abscission of daughter
cells (25,50). R6IP1 could be then another component of this
Rab11-driven machinery. Finally, it should be pointed out that
Rab11 and Rab6 could function at other stages of mitosis and
cytokinesis than those previously described. For instance,
Rab11 and several of its effectors can be found associated
with vesicles of unknown composition and function clustered
around the spindle poles after metaphase (38 and this study).
Rab6 (this study) and several effectors of Rab6, including
GAPCenA, also accumulate at the bridge during cytokinesis
(26; unpublished data). Thus, a tentative hypothesis is that
R6IP1 links the functions of Rab6 and Rab11 at metaphase
and during cytokinesis, as described in interphase.
Materials and Methods
Cloning of R6IP1
The partial cDNA for R6IP1 was originally identified from a yeast two-hybrid
screen of a mouse brain library using Rab6A Q22V as a bait [clone B; (27)].
The full-length cDNA was obtained by a combination of a new screen of
mouse brain l-ZAP2 library and a pseudo-RACE (rapid amplification of cDNA
ends) using a mouse brain cDNA library to get the 50 region of the cDNA.
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Oligonucleotides and siRNAs
The following oligonucleotides were synthesized using accession numbers NM_015312 for human R6IP1B and NM_021494.1 for mouse
R6IP1B: sense oligonucleotide on1 corresponding to nucleotides (nt)
292–311 in humans (AGTCGCTTCGCCGACTACTT) (the underlined C is
replaced by a T in mouse) and 277–296 in mouse upstream from the
insertion; anti-sense oligonucleotide on2 corresponding to nt 568–550 in
humans (CCAGCCCTTTCGGCATACA) and 553–535 in mouse
(CCAGCCCTTTGGGCATGCA); and anti-sense oligonucleotide on3 corresponding to nt 813–794 in humans (GGTCACAGGAGTGTCTTCAC) and nt
724–704 in mouse (GCATGTGGTAGAGGGTCTGCA). Two siRNAs for
R6IP1 were designed and produced by Qiagen. The target sequence of
siRNA R6IP1-1 is AGATGCAGTAGGAATGCTA and the one of siRNA
R6IP1-2 is CGAAAGGGCTGGCATTCAA. To knockdown Rab6A and A0 ,
we used the siRNA (siRab6A/A0 ) common for both Rab6 isoforms (23).
Rab11A siRNA is described in (51). Luciferase siRNA (Proligo) was used
as control.
Reverse transcription–polymerase chain reaction
Reverse transcription–polymerase chain reaction experiments were performed from mouse tissues (brain, leg muscle and small intestine), human
cell lines (HeLa; HTB13, derived from astrocytoma, and IMR32, derived
from neuroblastoma) and human tumor (Ewing tumors Erg and Fli, De Luca
rhabdoid tumor and neuroblastoma). Human materials were a generous gift
of Dr O. Delattre, Institut Curie, Paris. Total RNA and messenger RNA
(mRNA) were prepared by utilizing the Nucleospin RNA II kit (MachereyNagel) or the Fast Track Mag mRNA kit (Invitrogen), respectively. Messenger RNAs were then reverse transcribed using reverse transcriptase
(Finnzymes) and on3 oligonucleotide corresponding to human and mouse
R6IP1 transcripts. Subsequently, single-stranded cDNAs were amplified
using on1 and on2 oligonucleotides by PCR. The expected amplified cDNA
is 204 nt for R6IP1A or 276 nt for R6IP1B. Control plasmids encoding either
sequence were used as controls.
Cell transfection
Cells were trypsinized the day before transfection. For plasmid DNA
transfection, the calcium phosphate precipitate technique was used on
cells cultivated at about 70% confluency, using 2–4 mg total DNA in a well
of a 24-well plate. For double-stranded siRNA transfection, transfection
was performed using oligofectamine or HiPerFect reagent according to
the manufacturer’s (Qiagen) procedure, except that the siRNA transfection mix was applied to cells in suspension. Two microliters of
oligofectamine plus 0.2 mM siRNA in 250 mL total volume or 3 mL
HiPerFect plus 10 nM siRNA in 600 mL total volume were used per well
of 24-well plate. Oligofectamine mix was removed several hours before
cells were trypsinized the next day (day 1) and plated as needed. For
siRNA transfection followed by DNA transfection, 90% confluent, adherent cells were transfected with siRNA using Lipofectamine 2000 (day 0).
Cells were trypsinized the next day (day 1) and transfected again with
plasmid DNA using calcium phosphate precipitate (day 2). DNA transfection was usually stopped after 24 h.
Live cell imaging
Transfected cells were plated at day 1 in multiwell, uncoated glass dish,
and time-lapse sequences recorded at 10-min intervals as previously
described in Miserey-Lenkei et al. (26). For FLIM/FRET experiments, cells
were plated on 32- or 40-mm glass coverslip before transfection. During
observation, culture medium was replaced by Leibowitz medium (L15;
Gibco) for CO2 compensation and to avoid additional fluorescence from
the medium.
Two-photon FRET–FLIM experiments
The experimental setup is fully described elsewhere (52). Briefly, twophoton FRET/FLIM experiments were performed on a Leica SP2 AOBS
confocal scanner coupled to a femtosecond pulsed laser (Mira 900F;
Coherent Inc.). Lifetime measurements were performed using the timecorrelated single photon counting technique with Multi Channel Plate
1400
Photomultiplier Tube (MCP PMT) detector (R3809U-52; Hamamatsu) and
SPC 730 card (Becker&Hickl, Berlin, Germany). This setup allowed us to
measure the fluorescence decays for each pixel on an image.
Images were acquired by continuously scanning the samples with the laser
tuned at 830 nm for 300 seconds in order to get enough photons to build
a fluorescence lifetime image. Lifetime images were analyzed with SPC
IMAGE software (Becker&Hickl) by fitting data with either a single or a double
exponential function after fluorescence intensity threshold in order to avoid
pixels containing too few photons to achieve an adequate fitting. Mean
lifetime for the whole image was calculated from the distribution of values
corresponding to each pixel in the threshold image.
For each experimental condition, the mean lifetime was measured on
several cells, and the resulting distribution was filtered to decrease
measurement noise: all values corresponding to an occurrence frequency
lower than 5% of the maximum frequency were removed from the
analyzed data. The fluorescence lifetime histograms show the data for
n cells after this filtering and was calculated with bins of 100 picoseconds.
Mean fluorescence lifetime values and FRET efficiencies ! SD were
calculated by eliminating the cell population showing no FRET for cells
expressing Rab6 (A or A0 ), R6IP1 and Rab11A constructs (most left three
bars). FRET efficiencies were calculated from the fluorescence lifetimes
using the expression E ¼ 1 # t/t0, where t0 is the value of fluorescence
lifetime without acceptor.
Glutathione S-transferase pull-down and
immunoprecipitation experiments
These experiments were performed as described in Monier et al. (24). We
used, as a source of R6IP1, mouse brain PNS, R6IP1A from baculovirusinfected Sf9 cell lysates or purified R6IP1A from the same cells. Immunoprecipitation experiments were performed on HeLa cells transiently
expressing various R6IP1 constructs and Rab proteins. Usually, Rab
proteins were myc-tagged and R6IP1 were expressed in cells as GFP
chimera. After cotransfection with equivalent amount of both plasmids
(10 mg of each plasmid in a 10-cm-diameter dish), HeLa cells were lysed in
a buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2 and
0.5% Triton-X-100. Cell lysates were then centrifuged at 1000 $ g. 2.6 mg
of goat anti-Myc (Santa Cruz) was added together with protein G–
Sepharose. Immunoprecipitates were analyzed by SDS–PAGE, GFP fusion
proteins detected using an anti-GFP antibody (Roche) and myc-tagged
proteins detected using the goat anti-myc antibody.
Transferrin recycling
Cells transfected for 24 h with various GFP fusion proteins were incubated
for 1 h in fetal calf serum-free medium. They were then harvested using
Versen (Gibco BRL) at 1/25 and processed for Alexa 633–Tf binding and
recycling as described by Perez et al. (52). Briefly, cells were incubated for
1 h at 378C in the presence of 5 mg/mL Alexa 633–Tf (Molecular Probes) in
DMEM complemented with 10 mM HEPES pH 7.4 and 0.1% BSA. Cells
were then diluted in ice-cold PBS, pelleted and resuspended in cold DMEM
containing 10 mM HEPES pH 7.4, 10% BSA and 100 mg/mL unlabeled
holotransferrin (Sigma). One aliquot (t0) was removed in duplicate and
diluted in five volumes of ice-cold PBS containing 50 mg/mL unlabeled Tf
and incubated for about 10 min on ice, and cells were pelleted and fixed in
1% paraformaldehyde in Versen on ice. The rest of the cell suspension was
incubated at 378C with occasional stirring. Aliquots were removed at
various time-points until 60 min, and processed as t0. Fluorescenceactivated cell sorter (FACS) analysis was performed using a FACScalibur
as described previously (53).
Electron microscopy
Transfected HeLa cells were allowed to internalize Tf–HRP (US Biologicals)
or Tf–Biotin conjugates (Invitrogen) for 45 min and fixed with 2% (w/v)
paraformaldehyde with 0.2% (w/v) glutaraldehyde, in 0.1 M phosphate
Traffic 2007; 8: 1385–1403
Functional Characterization of Rab6IP1
buffer, pH 7.4. Cell pellets were processed for ultracryomicrotomy as
previously described (54). Ultrathin cryosections were prepared with an
ultracryomicrotome Ultracut FCS (Leica, Vienna, Austria), and single or
double immunogold labeled with the indicated primary antibodies and using
protein A conjugated to 10- or 15-nm gold [PAG10 and PAG15 (protein
A-colloidal gold)]. Sections were analyzed under a Philips CM120 electron
microscope (FEI, Eindhoven, the Netherlands), and digital acquisitions were
made with a numeric camera Keen View (Soft Imaging System, Munster,
Germany).
Others
R6IP1A was purified from Sf9 cells infected with baculovirus pFASTBac
encoding the full-length mouse R6IP1A protein. Two rabbit antisera were
raised against the protein (Eurogentech) and characterized by immunofluorescence and Western blotting. Immunofluorescence was performed as
previously described in Miserey-Lenkei et al. (26). For experiments involving round metaphasic cells, coverslips were coated with 1 mg/mL fibronectin (Chemicon) plus 0.0005% collagen (Sigma) prior to cell plating.
Endogenous Rab6 and Rab11 were revealed using previously described
specific antibodies (13,17) or using commercially available anti-Rab6 (Santa
Cruz) and anti-Rab11 (Zymed) antibodies.
endosomes/lysosomes) and accessibility to internalized Tf–HRP (endosomes). Tubulovesicular membranes that were located at the trans side
of the Golgi were considered as TGN. Endosomes were defined as
electron-lucent vacuoles with no internal membranes or few internal
vesicles and tubulovesicular elements closely apposed to the vacuoles.
MVBs (Multivesicular bodies) were compartments delimited by a membrane with numerous internal vesicles. Electron-dense compartments with
no or few internal membranes were classified as lysosomes. Other
vesicular structures dispersed in the cytoplasm with no assigned origin
were considered as ‘cytoplasmic vesicles’. Labeling of mitochondria with
anti-GFP antibodies was less than 0.1%. LAMP, Lysosomal-Associated
Membrane Protein; MVB, multivesicular bodies.
Figure S4: Localization of GFP–R6IP1A and endogenous Rab6 and
Rab11 in metaphase and cytokinesis. Cells were transfected for 24 h
with a plasmid encoding GFP–R6IP1A and then processed for immunofluorescence. Cells were labeled with antibodies against Rab6, Rab11 and
b-tubulin. In C, images were deconvolved for better visualization of Rab6positive structures in the intracellular bridge (arrowheads). Scale bars,
10 mm. Similar localization was found for GFP–R6IP1B (data not shown).
Table S1: Yeast two-hybrid assays
Acknowledgments
We thank Florence Niedergang for her help with FACS analysis, Yohanns
Bellaı̈che, Arnaud Echard and Joanne Young for critical reading of the
manuscript. We also thank Jean-Baptiste Sibarita and Vincent Fraisier,
members of the ‘RIO-Cell and Tissue Imaging Facility at Institut Curie’, for
their help in image acquisition and restoration processing. This work was
supported by the Institut Curie, the CNRS and the European Union (LSHMCT-2004-503228, Project acronyme ‘Signalling & Traffic’, http://www.
signallingtraffic.com).
Supplementary Materials
Figure S1: R6IP1 is a cytosolic protein. Post-nuclear supernatants were
prepared from mouse brain (lane a) and from HeLa cells (lane b). HeLa cell
PNS (lane c) was then centrifuged at 100 000 $ g in order to sort
membranes (mb, lane e) from the cytosol (lane d). Western blotting was
performed using a polyclonal rabbit antiserum generated against recombinant R6IP1A purified from baculovirus-infected cells. The antibody recognized in mouse PNS (lane a) a major band migrating at an apparent
molecular mass of 130 kDa, corresponding to the predicted size of the
protein. In HeLa cell PNS (lane b), the antibody stained additional bands.
They likely correspond to unrelated proteins because they were still present
after silencing of R6IP1 by siRNAs (data not shown). Molecular weight
markers are shown on the right.
Figure S2: Localization of the Golgi apparatus in CFP–Rab11A and
YFP–R6IP1A coexpressing cells. Cells transfected with CFP–Rab11A and
YFP–R6IP1A (corresponding to Figure 4C) were stained for endogenous
Rab6 (blue).
Figure S3: Quantification of subcellular localization of GFP–R6IP1A/B.
The relative distribution of R6IP1A and R6IP1B in transfected HeLa cells
was evaluated by analyzing directly under the electron microscope selected
cell profiles from two distinct grids. A total of 764 and 695 gold particles
were counted for R6IP1A and R6IP1B, respectively, and assigned to the
compartment over which they were located. The definition of the distinct
compartments was based on their morphology and their previous characterization by immunogold labeling with organelle markers (TGN46 for the
TGN and Lysosomal-Associated Membrane Protein-1 (LAMP-1) for late
Traffic 2007; 8: 1385–1403
Table S2: Statistics of Tf recycling experiments (Figure 9; p of Student’s
t-test)
Movies: HeLa cells treated with control (Movie S1) or R6IP1 siRNA (movie
S2) were analyzed for 72 h by time-lapse phase-contrast videomicroscopy.
Some R6IP1-siRNA-treated cells (Movie S2, arrow) entered mitosis normally,
rounded up, but were subsequently arrested in mitosis and died after several
hours. Details of a R6IP1-siRNA-treated cell undergoing a metaphase arrest
(Movie S3, arrow); note the alignment of chromosomes on the metaphase
plate during the metaphase block (arrowhead). Details of a cell (arrow)
undergoing cytokinesis defect after R6IP1 siRNA (Movie S4). The numbers
correspond to the time in minutes after the beginning of the recording.
Supplemental materials are available as part of the online article at http://
www.blackwell-synergy.com
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