BBRC
Biochemical and Biophysical Research Communications 312 (2003) 663–669
www.elsevier.com/locate/ybbrc
Reconstitution of vesicular transport to Rab11-positive
recycling endosomes in vitro
Rene Bartz, Corinne Benzing, and Oliver Ullrich*,1
Institut f€ur Biochemie, Universit€at Mainz, Becherweg 30, D-55128 Mainz, Germany
Received 20 October 2003
Abstract
Rab GTPases are key regulators of vesicular protein transport in both the endocytic and exocytic pathways. In endocytosis and
recycling, Rab11 plays a role in receptor recycling to plasma membrane via the pericentriolar recycling compartment. However, little
is known about the molecular requirements and partners that promote transport through Rab11-positive recycling endosomes.
Here, we report a novel approach to reconstitute transport to immunoabsorbed recycling endosomes in vitro. We show that
transport is temperature-, energy-, and time-dependent and requires the presence of Rab proteins, as it is inhibited by the Rabinteracting protein Rab GDP-dissociation inhibitor that removes Rab proteins from the membrane. Cytochalasin D, a drug that
blocks actin polymerization, inhibits the in vitro assay, suggesting that transport to recycling endosomes depends on an intact actin
cytoskeleton. Using an affinity chromatography approach we show the identification of Rab11-interacting proteins including actin
that stimulate transport to recycling endosomes in vitro.
Ó 2003 Elsevier Inc. All rights reserved.
Keywords: Rab11; Intracellular transport; Endosomes; Recycling; In vitro transport assay
Internalization of proteins and lipids in eukaryotic
cells occurs by receptor-mediated endocytosis [1]. Receptor-bound ligands are concentrated at the plasma
membrane, internalized into early (sorting) endosomes,
and are then transported to late endosomes and lysosomes for degradation. Most receptors, after separation
from the ligand, are recycled back to plasma membrane,
either directly from sorting endosomes or via the pericentriolar recycling endosome. Early, late, and recycling
endosomes are additionally involved in protein and lipid
exchange with the trans-Golgi network (TGN). These
transport steps are highly regulated implying a complex
process by a specific molecular machinery which regulates steps like vesicle formation, vesicle motility, and
specific docking/fusion of vesicles with the target membrane. Key regulators of transport are members of the
family of Rab GTPases that have been shown to localize
to specific subcellular compartments along the endocytic
*
Corresponding author. Fax: +49-40-42891-2681.
E-mail address: [email protected] (O. Ullrich).
1
Present Address: Hochschule f€
ur Angewandte Wissenschaften
Hamburg, Lohbr€
ugger Kirchstr. 65 D-21033 Hamburg, Germany.
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2003.10.172
and exocytic pathways, where they control distinct steps
of intracellular transport (for review see [2,3]). Therefore, Rab proteins have been used as compartmentspecific markers and tools for the characterization of
intracellular transport. In the endocytic pathway, Rab5
is localized to plasma membrane, clathrin-coated vesicles, and early endosomes, where it controls transport
from plasma membrane to sorting endosomes as well as
homotypic fusion of early endosomes. Rab11 has been
found mainly associated with pericentriolar recycling
endosomes regulating transport of internalized receptors
(like the transferrin receptor) back to plasma membrane
[4–6]. Overexpression of Rab11 mutants not only affects
the morphology of the recycling endosome, it also influences recycling of transferrin receptor through this
compartment [4]. Besides localizing to recycling endosomes, Rab11 has also been found on late Golgi and
post-Golgi membranes, suggesting an additional role in
transport from Golgi to plasma membrane [4,7–11] or in
recycling to Golgi [12].
Rab proteins execute their manifold functions in
formation, motility, and docking/fusion of vesicles
through interactions with various effector proteins, e.g.,
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R. Bartz et al. / Biochemical and Biophysical Research Communications 312 (2003) 663–669
by recruiting them to cellular membranes during protein
trafficking [2,3]. For Rab5 at least 20 cytosolic proteins
were recently identified as interacting factors [13]. The
Rab5 effector EEA1, for instance, is suggested to play a
role in homotypic docking of early endosomes because
of its two binding sites for Rab5. Vesicle motility may
also be regulated by Rab effectors. In this process, actin
and tubulin cytoskeleton serves as tracks for directed
movement of vesicles. Rab5 was shown to regulate
movement of early endosomes along microtubules
through an effector that is still unknown [14].
Much less is known about Rab11 effectors and their
precise function in regulation of transport through the
perinuclear recycling endosome. We have therefore established a novel in vitro assay, reconstituting transport
of transferrin receptor to recycling endosomes. This
assay is based on our previous biochemical approach to
analyze immunoisolated Rab11-positive recycling endosomes and Rab5-positive sorting endosomes [15]. In
our current study, we show that in vitro transport to
recycling endosomes is temperature-, ATP-, and timedependent and requires the presence of Rab proteins as
well as an intact actin cytoskeleton. We further
demonstrate the affinity purification of a fraction of
Rab11-interacting proteins that stimulates transport to
recycling endosomes.
Materials and methods
Materials. Polyclonal rabbit Rab11-antiserum was raised against
full-length recombinant His6 -Rab11 expressed in Escherichia coli [7]
and affinity purified essentially as previously described [16]. His6 RabGDI was expressed in E. coli and purified as described before [17].
Anti-actin antibody and cytochalasin D were purchased from Sigma
(Munich, Germany). Magnetic beads (Dynabeads M-450) were
obtained from Dynal (Hamburg, Germany). Reagents for the ATPregenerating and depletion system were purchased from Roche
Applied Science (Mannheim, Germany). All other reagents were purchased from Sigma, Amersham Biosciences (Freiburg, Germany), and
Gibco (Eggenheim, Germany).
Cell culture. Media and reagents for cell culture were obtained from
Gibco (Eggenheim, Germany). Chinese hamster ovary (CHO) cells
were kindly provided by M. Zerial (Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany) and were grown
in Ham’s F12 containing 10% (v/v) heat-inactivated fetal bovine serum
(FBS), 100 U/ml penicillin, 100 lg/ml streptomycin, and 2 mM
L -glutamine.
Acridinium ester labeling of transferrin (Ac-Tfn). Acridinium C2
NHS ester (Biotrend, Cologne, Germany) was covalently bound to
human holo-transferrin (Sigma, Munich, Germany) and purified on a
PD-10 column (Pharmacia, Freiburg, Germany) according to the
manufacturer’s protocol. Coupled transferrin was finally dialyzed
against an iron saturation buffer (250 mM Tris, pH 8.2, 250 mM FeCl3 ,
1 mM NaHCO3 , and 2 mM nitrilotriacetic acid) to ensure that transferrin is iron-loaded, followed by dialysis against PBS.
Preparation of endosome-enriched fractions. Preparation of early
and recycling endosome-enriched fractions using a step-flotation gradient was performed by a slightly modified protocol that was first
described by Gorvel et al. [18]. Briefly, CHO cells were grown on
10 cm2 dishes, washed with PBS, and scraped with a rubber policeman.
Cells were homogenized in homogenization buffer (250 mM sucrose,
3 mM imidazole, pH 7.4) and post-nuclear supernatants (PNSs) were
prepared. PNS was adjusted to 40.6% sucrose, loaded into a centrifugation tube, then overlaid with 12 ml of 35% sucrose, 3 mM imidazole, pH 7.4, followed by 8 ml of 25% sucrose, 3 mM imidazole, pH
7.4, and finally filled up with homogenization buffer. Step gradients
were centrifuged at 108,000g for 3 h at 4 °C. The endosome-enriched
35%/25% sucrose interphase was collected using a fractionator (Auto
Densi-Flow, Labconco, Kansas City, USA).
For the preparation of acridinium–transferrin (Ac-Tfn)-labeled
endosomes, cells were washed twice with serum-free Ham’s F12 at
room temperature and then depleted from serum transferrin by incubation with serum-free Ham’s F12 for 1 h at 37 °C and 5% CO2 ,
followed by labeling with serum-free Ham’s F12 containing iron-saturated Ac-Tfn (20 lg/ml) at 37 °C and 5% CO2 for 30 min. To remove
cell surface-bound Ac-Tfn, cells were washed twice at 4 °C with PBS,
PBS/0.5% bovine serum albumin (BSA), and PBS. For preparation of
endosome-enriched fractions, cells were processed as described above.
Preparation of anti-Rab11-coated magnetic beads for immunoadsorption. To prepare anti-Rab11-coated magnetic beads, affinity
purified goat anti-rabbit IgG (Fc-specific) from Dianova (Hamburg,
Germany) was coupled to p-toluene sulfonylchloride-activated Dynabeads M-450 by incubation of 10 lg/mg beads in 0.1 M borate buffer,
pH 9.5, for 24 h at room temperature. Beads were then blocked and
washed according to the manufacturer’s instructions. The amount of
bound IgG was >80%. Finally, beads were incubated with affinity
purified rabbit anti-Rab11 in PBS/0.5% BSA for 12 h at 4 °C, followed
by three washes in PBS/0.1% BSA.
Immunoadsorption and in vitro fusion assay. For immunoisolation
of acceptor recycling endosomes, anti-Rab11-coated magnetic beads
were incubated with the unlabeled endosome-enriched fraction (35%/
25% sucrose interphase from the step-flotation gradient) at 60–80 lg
protein/10 mg beads diluted in PBS/0.1% BSA at 4 °C for 4 h rotating
end-over-end. Beads were then collected with a magnet and then resuspended in fusion buffer (12 mM Hepes, 75 mM potassium acetate,
and 1.5 mM magnesium acetate, pH 7.3) containing 0.1% BSA. For in
vitro fusion, beads were collected with a magnet on ice, the supernatant was removed, and immunoisolated unlabeled endosomes were
incubated at 37 °C with Ac-Tfn-labeled donor endosomes in fusion
buffer supplemented with 10 mg/ml rat liver cytosol and an ATP-regenerating system (1 mM ATP, 8 mM creatine phosphate, and 40 lg/ml
creatine phosphokinase). After incubation, beads were collected and
washed at 4 °C with 300 mM KCl/0.1% BSA followed by washing with
PBS. To determine fusion of Ac-Tfn-labeled donor endosomes with
immunoisolated unlabeled acceptor endosomes, membranes associated
with beads were solubilized with PBS/0.5% Triton X-100 and flashlight
chemiluminescence was measured in acridinium trigger solution (Biotrend, Cologne, Germany) for 2 s using a Berthold Luminometer LB
9501.
Affinity purification of Rab11-interacting proteins. Forty milligrams
recombinant His6 -Rab11 was coupled to 10 ml CNBr–Sepharose 4B
beads according to the manufacturer’s instructions (Pharmacia, Freiburg, Germany). Rat liver cytosol was obtained by homogenizing rat
livers in a Potter–Elvehjem in homogenization buffer (250 mM sucrose, 3 mM imidazole, pH 7.4) and centrifugation of the homogenate
at 15,000g for 10 min at 4 °C. The supernatant was passed through a
sterile gauze and the filtrate was centrifuged at 200,000g at 4 °C for
1 h. Supernatant was diluted to 10 mg/ml by adding one volume of 2
incubation buffer (40 mM Hepes, 150 mM potassium acetate, 3 mM
magnesium acetate, and 2 mM DTT, pH 7.3) and incubated with
Rab11-beads in batch for 1 h at 4 °C under slow rotation. Beads were
transferred to a column and washed with 20 bed volumes of washing
buffer (20 mM Hepes, 75 mM potassium acetate, 1.5 mM magnesium
acetate, and 1 mM DTT, pH 7.3) followed by one wash with 20 bed
volumes of high salt buffer (same buffer containing 250 mM NaCl).
Bound proteins were eluted with one bead volume of elution buffer
(20 mM Hepes, 75 mM potassium acetate, 1.5 mM magnesium acetate,
R. Bartz et al. / Biochemical and Biophysical Research Communications 312 (2003) 663–669
3 M NaCl, and 1 mM DTT, pH 7.3). Eluate was precipitated with 20%
TCA for 1 h on ice. After spinning at 14,000g for 20 min at 4 °C,
supernatant was discarded and pellet was dissolved in 2 SDS-sample
buffer containing 2% of 2-mercaptoethanol. Samples were boiled and
analyzed by SDS–PAGE and Western blotting. To identify also
strong interacting proteins that were not eluted, beads were boiled
in SDS-sample buffer and loaded on SDS–PAGE. For testing the
eluates in the fusion assay, eluted proteins were dialyzed against
fusion buffer.
Results
Characterization of transport to recycling endosomes
in vitro
To investigate transport to Rab11-positive recycling
endosomes we have developed an in vitro transport assay based on a recently established protocol for the
immunoisolation of sorting and recycling endosomes
using Rab proteins as compartment specific markers
[15]. Isolated Rab5-positive sorting endosomes and
Rab11-positive recycling endosomes differed in protein
composition as well as morphology and were sequentially encountered by endocytosed transferrin.
Here, in our novel transport assay, recycling endosomes immunoisolated with anti-Rab11-beads from
unlabeled CHO cells were used as the acceptor fraction
(Fig. 1). Beads-bound acceptor membranes were then
incubated with donor endosomes labeled with acridinium–transferrin (Ac-Tfn) from cells that had internalized this endocytic marker. The fusion reaction was
665
performed in the presence of cytosol and an ATP-regenerating system under various conditions (temperature, ATP-depletion, time, different factors, and drugs).
Transport to recycling endosomes was defined as
transport of Ac-Tfn from the donor fraction to the
immunoisolated acceptor fraction as measured by
flashlight chemiluminescence using a luminometer (see
Materials and methods).
First, control experiments were performed to determine the specificity of the transport reaction. To exclude
unspecific binding of labeled endosomes to magnetic
beads, control beads without anti-Rab11 antibodies
(IgG control) were used in parallel. Our results show
efficient transport to anti-Rab11 coupled beads, while
only little unspecific binding (7%) of donor endosomes
was observed for control beads (Fig. 2A). To control for
this minor unspecific binding all our experiments were
performed with control beads lacking anti-Rab11 antibody (IgG control). The values from unspecific binding
(IgG control) were subtracted from the values obtained
with anti-Rab11 beads and calculated as percentage of
maximum fusion. For a further characterization of the
in vitro transport assay we analyzed the role of temperature and ATP for transport to recycling endosomes.
In living cells, intracellular transport is inhibited
strongly at 4 °C. Consistent with the in vivo data, in our
in vitro assay transport was inhibited by approx. 90% in
comparison to 37 °C (Fig. 2B). Then, in order to investigate the ATP-dependence of transport, we depleted
cytosolic ATP using an ATP-depletion system. Our results show an inhibition of 50% after depletion of cytosolic ATP (Fig. 2C), reflecting the general requirement
of ATP for membrane transport. Next, we investigated
in vitro fusion for different periods of time. We observed
that transport of Ac-Tfn to immunoisolated recycling
endosomes first increased in a time-dependent manner
and then reached a plateau at 30 min (Fig. 3A). Altogether, our results from the novel in vitro assay demonstrate that transport to recycling endosomes is
temperature-, ATP-, and time-dependent, requirements
that are comparable to those of other membrane
transport assays.
Transport to recycling endosomes requires Rab proteins
and an intact actin cytoskeleton
Fig. 1. In vitro assay to study endocytic traffic to recycling endosomes.
The assay is based on fusion of acridinium–transferrin (*Tfn)-labeled
endosomes (donor) with immunoisolated Rab11-positive recycling
endosomes (acceptor). Acceptor endosomes are prepared from unlabeled CHO cells by immunoisolation of Rab11-positive recycling endosomes from an endosome-enriched fraction. Donor endosomes
represent an endosome-enriched fraction obtained from cells labeled
with *Tfn for 30 min at 37 °C. For the fusion reaction, donor endosomes are incubated with beads-bound acceptor endosomes in the
presence of cytosol and an ATP-regenerating system at 37 °C. Beads
are then washed thoroughly and fusion is analyzed by the amount of
*Tfn transferred to beads-bound recycling endosomes, as measured by
Flashlight-luminescence using a luminometer.
Rab function requires the activity of additional Rabinteracting proteins that assist in the formation, transport, and docking/fusion of transport vesicles [2,3]. An
important accessory factor is Rab-GDP-dissociation
inhibitor (RabGDI) that extracts Rab proteins from
membranes in their GDP-bound form, allowing their
return to donor membranes, which is essential for
multiple rounds of vesicle transport. Because of this
ability RabGDI has been extensively used as a tool for
studying Rab function in vivo and in vitro [9,19–25]. For
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Fig. 2. Transport to recycling endosomes in vitro is specific and inhibited by low temperature and ATP-depletion. (A) Beads coated with anti-Rab11antibodies (anti-Rab11) or with anti-rabbit antibodies (IgG control) were used for immunoisolation of acceptor recycling endosomes. Beads were
then incubated with donor endosomes for 30 min at 37 °C. (B) Anti-Rab11-beads with immunoisolated acceptor membranes were incubated with
donor endosomes for 30 min at 37 or 4 °C. (C) Acceptor membranes immunoisolated with anti-Rab11 were incubated for 30 min at 37 °C with donor
endosomes in the presence of cytosol and an ATP-regenerating system (control) or an ATP-depletion system (ATP-depletion). Samples were processed as described in Fig. 1. Here the mean of three experiments is shown and the error bars represent the standard deviation.
Fig. 3. In vitro transport to immunoisolated recycling endosomes is time-dependent and inhibited by RabGDI and cytochalasin D. (A) Immunoisolated Rab11-positive recycling endosomes were incubated with donor endosomes at 37 °C for increasing periods of time and processed as
described in Fig. 1. (B,C) Recycling endosomes immunoisolated with anti-Rab11-antibodies were incubated with donor endosomes for 30 min at
37 °C in the presence or absence (control) of (B) 10 lM RabGDI or (C) 20 lM cytochalasin D and processed as described in Fig. 1. The mean of three
experiments is shown and the error bars represent the standard deviation.
transport to recycling endosomes in vitro, the addition
of 10 lM RabGDI to the reaction mixture reduced
transport to recycling endosomes to approx. 50% in
comparison to the control (Fig. 3B). This result suggests
that Rab proteins extracted from endosomal membranes
by RabGDI are required for efficient transport to recycling endosomes.
Vesicular transport involves elements of the cytoskeletal system by serving as tracks for directed movement of vesicles and recent studies have implicated Rab
proteins in this movement [26,27]. Rab27a regulates the
movement of melanin containing vesicles by interacting
with the actin motor protein myosin Va [28] and Rab11
was found to interact with myosin Vb [29]. To investigate the role of actin cytoskeleton in transport to recycling endosomes in vitro, we tested the effect of
cytochalasin D that leads to the depolymerization of
actin filaments. Addition of the drug to the assay system
reduced transport to approx. 40% (Fig. 3C), suggesting
a role of actin filaments in transport to recycling endosomes. This finding is supported by our previous results,
demonstrating the enrichment of actin on immunoisolated Rab11-positive recycling endosomes compared to
Rab5-positive sorting endosomes [15].
R. Bartz et al. / Biochemical and Biophysical Research Communications 312 (2003) 663–669
Identification of Rab11-interacting proteins that stimulate
transport to recycling endosomes
Since actin appears to play an important role in
transport to recycling endosomes, we next investigated
whether this effect might be mediated through an interaction with Rab11. We therefore used an affinity
chromatography approach to search for possible
Rab11-binding molecules as actin and other potential
interacting proteins. Purified recombinant His6 -Rab11
was immobilized to CNBr–Sepharose beads and incubated with rat liver cytosol at 4 °C. Beads were then
extensively washed and proteins were eluted under highsalt conditions. Eluted fractions were TCA-precipitated
and analyzed by SDS–PAGE followed by silver staining
and Western blotting. To analyze also the proteins remaining bound to Rab11-beads even after high salt
elution, beads were boiled in SDS-sample buffer. Several
protein bands could be detected after SDS–PAGE and
silver staining in the eluate of Rab11-beads but not in
that of control beads lacking Rab11 (Fig. 4A). Among
these proteins actin was identified by Western blotting,
while no actin was found in control beads (Fig. 4B).
Actin could be partially eluted from the beads, however,
667
most actin remained strongly bound to Rab11-beads,
even after high salt incubation. These results demonstrate that a number of cytosolic proteins bind directly
or via additional factors to Rab11.
Rab-interacting proteins were shown to stimulate or
inhibit particular steps of protein transport in vivo and
in vitro [2,3]. We therefore analyzed whether the Rab11interacting proteins from the affinity chromatography
would affect transport to Rab11-positive recycling endosomes. The salt-extracted protein fraction was first
dialyzed against fusion buffer and then tested in our
transport assay. Indeed, there was a clear stimulation of
transport of approx. 40% by the fraction (Fig. 4C), indicating the presence of Rab11-binding factors that are
involved in the regulation of trafficking to recycling
endosomes.
Discussion
Although uptake and recycling of transferrin has
been studied extensively in vivo, relatively little is known
about the underlying molecular mechanisms and molecules that regulate transport through the recycling
Fig. 4. Cytosolic proteins affinity purified with immobilized His6 -Rab11 contain actin and stimulate transport to recycling endosomes in vitro. (A,B)
His6 -Rab11 was expressed in E. coli, purified, and covalently bound to CNBr-activated Sepharose 4B. His6 -Rab11-beads or control beads (minus
His6 -Rab11) were incubated with rat liver cytosol for 1 h at 4 °C, beads were extensively washed, and bound proteins were eluted with high salt buffer.
Cytosol (cytosol), beads after elution boiled in SDS-sample buffer (boiled beads), and eluted proteins (eluate) were separated by SDS–PAGE and
analyzed by (A) silver staining or (B) Western blotting using antibodies against actin. (C) The eluted fractions from control beads and His6 -Rab11beads were dialyzed against fusion buffer and added to a standard fusion reaction mix (30 min at 37 °C). Here the mean of three experiments is shown
and the error bars represent the standard deviation.
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R. Bartz et al. / Biochemical and Biophysical Research Communications 312 (2003) 663–669
endosome. A regulator of the recycling pathway is
Rab11 and its function(s) depend(s) on the activity of
probably a large number of Rab11-interacting proteins.
Although some of them have been identified only little is
known about their function [29–36]. Therefore, if one
assumes for Rab11 a similar number of accessory factors as they were found for Rab5 [13], then we have to
expect additional Rab11-binding molecules to be found
supporting additional Rab functions.
To address these questions in vitro, we have established a novel cell-free assay to reconstitute endosomal
transport to recycling endosomes. Donor endosomes
labeled with Ac-Tfn are mixed with Rab11-positive
immunoisolated recycling (acceptor) endosomes in buffer containing cytosol and an ATP-regenerating system.
After the fusion reaction at 37 °C, beads-bound acceptor
endosomes are washed extensively and transferred
Ac-Tfn is measured using a luminometer.
Our results show that the assay exhibits the typical
criteria of an in vitro system to study membrane transport. First, transport is specific, since efficient transfer of
Ac-Tfn from donor endosomes to beads only occurred
when anti-Rab11-immunoisolated recycling endosomes
were present, but not to control beads. Second,
transport was clearly temperature-dependent as it
was strongly inhibited at 4 °C. Third, there was an
ATP-dependency of transport. In the presence of an
ATP-depletion system, transfer of Ac-Tfn to acceptor
recycling endosomes was significantly inhibited.
While low temperature strongly reduced transport as
observed in vivo, the removal of ATP resulted only in a
partial (50%) inhibition of transport. Our finding is
consistent with other transport assays, where no complete inhibition of transport was found in the absence of
ATP. This suggests that some steps of the multiple step
process of fusion might be ATP-dependent while others
are not. When the fusion reaction is initiated, a proportion of endosomes could be already primed for
fusion and beyond the energy-dependent step.
Fusion of donor and acceptor endosomes exhibited a
time-dependent increase reaching a plateau at 30 min of
time. The plateau in this time course is likely to reflect
quantitative fusion of fusion-competent endosomes as
well as depletion of rate limiting factors from cytosol
that are essential for docking and fusion.
Membrane transport depends on the presence of
regulatory Rab proteins [2,3]. We therefore added
RabGDI to the transport assay as a well-established
inhibitor of Rab function based on the ability of RabGDI to extract Rab proteins in their GDP-bound state
from membranes [9,19–23]. RabGDI reduced transport
to approx. 50% suggesting a Rab-mediated regulation of
transport to recycling endosomes. Consistent with other
transport assays [19,37], inhibition of transport by
RabGDI was only partial, probably because of already
primed endosomes that contain Rab proteins in their
active GTP-bound form as well as other proteins needed
for fusion. These GTP-bound Rab proteins would not
be extracted by RabGDI from their membranes and
would stimulate directly or indirectly vesicular fusion.
Recently, we have shown by immunoisolation that
actin is present predominantly on Rab11-positive
recycling endosomes, while little actin was found on
Rab5-positive sorting endosomes [15]. Actin has been
functionally linked to the cellular localization of organelles as well as vesicular transport. The movement of
vesicles along the actin cytoskeleton is powered by
members of the myosin protein family. Using a yeast
two-hybrid screening an interaction of Rab11 with the
actin motor myosin Vb was detected, suggesting a role
of Rab11 in actin mediated transport to recycling endosomes [29]. Our data presented here also support an
involvement of the actin cytoskeleton in intracellular
transport to perinuclear recycling endosomes, possibly
by interacting directly or via an accessory factor with
Rab11. First, cytochalasin D, which blocks actin polymerization, inhibited trafficking of Ac-Tfn to recycling
endosomes in vitro. Second, among other proteins, actin
interacted strongly with immobilized His6 -Rab11 and
the eluted protein fraction stimulated transport to
Rab11-positive recycling endosomes in vitro. Although
the isolation of Rab11-binding factors that stimulate this
transport step is very encouraging we cannot yet define
the identities of this activity. It could be actin and actinbinding proteins, however, also other Rab11 effectors
might be present in this fraction. Our future goal will
therefore be to further analyze the fraction of Rab11interacting proteins in order to identify new molecular
components that affect membrane transport along the
recycling pathway. The cell-free system described in this
study should greatly facilitate this analysis.
Acknowledgments
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to O.U.). R.B. was supported by a graduate
scholarship of the University of Mainz (LGFG).
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