Regulation of early endocytic vesicle motility and fission in a reconstituted system

Eustratios Bananis1,2, John W. Murray1,2, Richard J. Stockert1, Peter Satir1,2 and Allan W. Wolkoff1,2,*

1 Marion Bessin Liver Research Center
2 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

* Author for correspondence (e-mail: wolkoff{at}aecom.yu.edu)

Accepted 14 March 2003


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 Materials and Methods
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We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-{gamma}-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-{gamma}-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-{gamma}-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

Key words: Endocytosis, Microtubules, Rabs, Kinesin, Motility


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Receptor-mediated endocytosis is a process in which ligands bind to specific cell surface receptors and are internalized within clathrin-coated pits whose constriction leads to coated vesicle formation. Clathrin is released rapidly, and uncoated vesicles mature into early/sorting endosomes (Marsh and McMahon, 1999Go; Mellman, 1996Go; Mukherjee et al., 1997Go). Acidification of early endosomes (Forgac, 1998Go) leads to dissociation of most receptor-ligand complexes (Harford et al., 1983aGo; Harford et al., 1983bGo). Subsequently, early endocytic vesicles undergo a series of fissions resulting in the segregation of receptor from ligand (Bananis et al., 2000Go; Mellman, 1996Go; Mukherjee et al., 1997Go; Wolkoff et al., 1984Go). Ligands destined for degradation traffic within late endosomes to lysosomes (Harford et al., 1983bGo; Mukherjee et al., 1997Go; Wolkoff et al., 1984Go) while receptor-enriched vesicles are recycled back to the cell surface. Although the processes associated with coating and uncoating of the early endosome have received significant attention, little is known regarding the mechanism of endocytic vesicle fission that results in receptor-ligand segregation. In previous studies (Bananis et al., 2000Go; Murray et al., 2000Go), we prepared early endocytic vesicles from rat liver 5 minutes after intravenous injection of Texas-Red-labeled asialoorosomucoid (ASOR). We showed that these vesicles are presegregation vesicles that contain both ligand and receptor (Bananis et al., 2000Go). Using a cell-free fluorescence microscopy system, we were able to reconstitute microtubule (MT)-based motility and fission of these vesicles and showed that both plus- and minus-end-directed kinesins, but not dynein, are required for these processes (Bananis et al., 2000Go). An important question is whether segregation of endocytic vesicles is a regulated process, and whether the potential regulatory apparatus is functional in the purified vesicles used in our assays.

Studies by other laboratories in different endocytic systems have implicated several Rab proteins as regulatory factors (Mohrmann and van der Sluijs, 1999Go; Rodman and Wandinger-Ness, 2000Go; Zerial and McBride, 2001Go). In the present study, using immunofluorescence microscopy, image analysis, and biochemical assays, we provide evidence that Rab4 in the hepatocyte ASOR/asialoglycoprotein receptor (ASGPR) system is a regulator of early endocytic vesicle processing, serving as a `molecular switch' (Zerial and McBride, 2001Go) that, in its GTP-bound state, interacts with vesicle-associated proteins to inhibit vesicle motility. Addition of GDP converts Rab4 to its GDP-bound state and results in activation of motility and vesicle fission, corresponding to increased ligand-receptor vesicular sorting.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemicals and reagents
Asialoorosomucoid (ASOR) was prepared from human orosomucoid (Sigma) by acid hydrolysis (Stockert et al., 1980Go). All nucleotides and their analogues were purchased from Sigma (St Louis, MO). GTP-{gamma}-[35S] (1250 Ci/mmol) and [3H]GDP (25-50 Ci/mmol) were purchased from NEN (Boston, MA). Mouse IgM monoclonal antibodies against the dynein intermediate chain and the kinesin I heavy chain were obtained from Sigma. Mouse IgG monoclonal antibodies against the amino terminal of the dynein intermediate chain and the kinesin I heavy chain were obtained from Chemicon International (Temecula, CA). Mouse IgG monoclonal antibody against Rab-GDI was obtained from Synaptic Systems (Gottingen, Germany). Affinity purified rabbit IgG against KIFC2 was purchased from Affinity Bioreagents (Golden, CO). Affinity purified rabbit IgG was prepared against a KLH-linked peptide (VNRWACERKRDITYC) corresponding to a sequence on the cytoplasmic tail of the rat asialoglycoprotein receptor (ASGPR) H2 subunit (Stockert, 1995Go). Mouse monoclonal antibodies against Rab4, Rab5, Rab11, dynamin II, EEA1 and dynactin p50 were obtained from Transduction Laboratories (San Diego, CA). Affinity purified rabbit IgG against Rab7 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antiserum against clathrin heavy chain was kindly provided by Dennis Shields (Albert Einstein College of Medicine, Bronx, NY). Affinity purified rabbit IgG against dynamin II was a kind gift from Mark McNiven (Mayo Clinic, Rochester, MN). Purified goat antibody against GST was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The connexin 43 C-terminus (aa 255-382) was generated as a GST fusion protein and released from GST using Precission protease (Amersham) and purified and concentrated (Duffy et al., 2001Go). All other reagents were from Sigma unless otherwise noted.

Assay of early endocytic vesicle motility and fission in vitro
Texas-Red-labeled early endocytic vesicles were prepared from livers that were removed from 200-250 g male Sprague-Dawley rats (Taconic Farms, Germantown, NY) 5 minutes after portal venous injection of Texas-Red-labeled ASOR (Bananis et al., 2000Go; Murray et al., 2000Go). All animal procedures were approved by the University Animal Use Committee. After Dounce homogenization, a postnuclear supernatant was prepared and chromatographed on a Sephacryl S200 column. Vesicle-enriched peaks were pooled and subjected to centrifugation (200,000 g for 135 minutes) on a sucrose step gradient consisting of 1.4, 1.2 and 0.25 M sucrose. Vesicles were harvested from the 1.2 M/0.25 M sucrose interface and stored at -80°C until used. Details of these procedures have been published recently (Murray et al., 2000Go). Three vesicle isolations were used for the majority of the data presented. Significant variation between isolations was not observed. Motility assays were performed in a chamber consisting of two pieces of double-sided tape sandwiched between optical glass; the internal volume was approximately 3 µl. The chamber was coated with 0.02 mg/ml DEAE-dextran (Amersham Pharmacia Biotech, Piscataway, NJ) and rhodamine-labeled, Taxol-stabilized microtubules (MTs) were added and incubated for 3 minutes (Bananis et al., 2000Go). Following three washes with PMEE motility buffer (35 mM Pipes, 5 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA, 4 mM DTT, 20 µM Taxol, 2 mg/ml BSA, pH 7.4, containing an oxygen scavenging system) containing 5 mg/ml casein, endocytic vesicles were added to the chamber, incubated for 10 minutes, and washed. Motility was initiated by the addition of 50 µM ATP in the absence of a regenerating system. In some experiments, polarity-marked rhodamine-labeled microtubules were prepared to determine directionality of moving Texas-Red–ASOR vesicles (Murray et al., 2000Go). In other experiments, MT-bound vesicles were incubated with 4 mM guanine nucleotide analogues, 2 µM (100 mg/ml) purified GST-Rab4 or GST-Rab5 proteins, or 0.5 µM (0.030 mg/ml) purified rat liver Rab-GDI for 5 minutes at room temperature, prior to ATP addition.

Image analysis
Imaging was performed at the Analytical Imaging Facility of the Albert Einstein College of Medicine. A 60x, 1.4 numerical aperture planapo objective was used on an Olympus 1X70 inverted microscope containing automatic excitation and emission filter wheels connected to a Photometrics charge-coupled device camera run by IPLab Spectrum software (Scanalitics, Fairfax, VA) running on a Power Macintosh. IPLab Spectrum scripting software was used to collect images rapidly and to switch between fluorescence channels. Images were also recorded directly onto videotape. In all experiments the microscope stage was maintained at 35°C. Videos were digitized with the use of the Scion Image (Scion Corporation, Frederick, MD) movie-making macro (1 frame/second) and saved as tiff files. Corresponding frames indicating the staining in the different fluorescence channels were merged using Adobe Photoshop 5.0.

Purification of Rab-GDI from rat liver
Rab-GDI was isolated as previously described (Sasaki et al., 1990Go) with modifications. Rat liver was used as the source material. Volumes were scaled down 15-fold, and Rab-GDI was released from the final Mono Q column with 33 mM KCl (in appropriate buffer) rather than extensive washing. Rab-GDI was detected as a doublet at the appropriate molecular weight by immunoblot and Coomassie-blue SDS-Page. The purified Rab-GDI did not contain Rab4 as demonstrated by immunoblotting with anti-Rab4.

Immunoblot analysis
Appropriate protein samples (~15 µg total protein) were subjected to 10-20% gradient SDS-Page under reducing conditions. Following transfer to nitrocellulose, the blot was blocked in PBS containing 0.1% Tween 20 and 10% nonfat dried milk. Immunoblot analysis was then performed using appropriate primary and HRP-conjugated secondary antibodies as we have described previously (Hilgard et al., 2002Go).

Preparation of Rab4 and Rab5 GST-fusion proteins
Rab4 and Rab5 cDNAs in pBlueScriptKS+ were a gift from Ira Mellman (Yale University School of Medicine, New Haven, CT). Rab4 and Rab5 cDNAs were digested with EcoRI and inserted in frame into pGEX-6P-2 and pGEX-6P-1 vectors, respectively (Amersham Pharmacia Biotech). Bacterial expression of GST fusion proteins was induced with 0.5 mM IPTG for 3.5 hours at 37°C. Bacteria were lysed by sonication, and recombinant fusion proteins were subjected to affinity chromatography on GSH-agarose from which they were eluted with 5 mM GSH. Recovery of GST-Rab4 and GST-Rab5 was determined by immunoblotting with goat anti-GST antibody, as well as with specific Rab4 and Rab5 monoclonal antibodies.

Immunocolocalization of vesicle-associated proteins with fluorescent early endocytic vesicles
Endocytic vesicles were attached to motility chamber-bound microtubules (Bananis et al., 2000Go). Primary antibodies diluted as appropriate in 5 mg/ml casein-containing PMEE buffer were perfused through the vesicle-containing motility chamber, incubated for 6 minutes, and blocked with motility buffer containing 5 mg/ml casein. Cy2- or FITC-labeled affinity-purified secondary antibody was added and incubation was continued for an additional 6 minutes. After extensive washing with PMEE buffer containing 5 mg/ml casein, immunofluorescence microscopy was performed. In some experiments, prior to primary antibody perfusion, MT-bound vesicles were incubated with guanosine nucleotide analogues with or without 2 µM GST-Rab4, 2 µM GST-Rab5, or 0.5 µM Rab-GDI for 5 minutes and washed with motility buffer.

Assay of dissociation of Rab4-GDP from endocytic vesicles in vitro mediated by Rab-GDI or monoclonal antibody to Rab4
Endocytic vesicles (40 µg total protein in ~50 µl) were reconstituted with 120 µl PMEE buffer containing 2 mg/ml BSA and were centrifuged at 15 psi for 5 minutes at room temperature in a Beckman Airfuge (Palo Alto, CA). Pellets were reconstituted in 50 µl PMEE buffer or in PMEE buffer containing 4 mM GDP or 4 mM GTP-{gamma}-S and were incubated at 37°C for 15 minutes. Following an additional centrifugation step, vesicle pellets were reconstituted in 50 µl buffer containing 1 µM Rab-GDI and/or 4 mM GDP or 4 mM GTP-{gamma}-S. Vesicles were incubated at 37°C for 15 minutes and pelleted at 15 psi for 5 minutes. Supernatants and pellets were subjected to 4-20% gradient SDS-Page under non-reducing conditions and immunoblot analysis with monoclonal antibody to Rab4. Densitometric analysis was performed using a Fluorchem imaging system (Alpha Innotech, San Leandro, CA). Similar conditions were used to examine Rab4 monoclonal antibody-mediated dissociation of Rab4-GDP from endocytic vesicles. In these studies, vesicles were incubated with buffer containing 0.05 µM (0.75 mg/ml) Rab4 IgG and/or 4 mM GDP following the second centrifugation step.

Binding of GST-Rab4 to endocytic vesicles in vitro
Vesicles (60 µg total protein in ~70 µl) were reconstituted with 120 µl PMEE buffer containing 2 mg/ml BSA and were centrifuged at 15 psi for 5 minutes at room temperature. Pellets were reconstituted in 70 µl buffer containing 0.5 µM GST-Rab4 and were incubated at 37°C for 15 minutes. Following three additional centrifugation and wash steps with 150 µl buffer, vesicle pellets were subjected to 4-20% gradient SDS-Page and immunoblot analysis with Rab4 monoclonal antibody.

Surface plasmon resonance studies of ligand-antibody binding
An IAsys manual biosensor (Affinity Sensors, Cambridge, UK) using surface plasmon resonance technology was employed to quantify the binding of antibodies to their ligands at 25°C (Duffy et al., 2001Go). GST-Rab4 and GST-Rab5 as well as Rab4 and Rab5 monoclonal antibodies were dialyzed into PBS-T (10 mM sodium phosphate, 2.7 mM potassium chloride, 138 mM sodium chloride, 0.05% Tween 20). GST-Rab4 and GST-Rab5 were crosslinked to carboxymethyl dextran cuvettes using NHS/EDC activation according to the manufacturer's protocols. Antibody was added to GST-Rab4 or GST-Rab5 coated cuvettes at the indicated concentrations and instrument response (in arc seconds) was recorded for 10 minutes. After this time, cuvettes were rinsed and the surface regenerated (bound protein removed) by incubating in 50 mM HCl.

The equilibrium constant for antibody-ligand interaction was estimated by plotting the maximal binding response (extrapolated to infinite time) versus the total concentration of antibody and fitting this to the quadratic equation:

using the coefficients a=-1, b=Kd+Lt+Abt, c=LtxAbt, where Kd is the bimolecular dissociation constant, Lt is the total concentration of ligand bound to the cuvette (i.e. GST-Rab4 or -Rab5), accessible to antibody, and Abt is the total concentration of antibody.

Alternatively, the free concentration of antibody was calculated by subtracting the bound from total and plotting free versus bound to determine the apparent Kd in standard Langmuir binding isotherm fashion. A calibration constant of 50 arc s ng-1 mm2 was used to convert response into nanograms of protein bound (Hall and Winzor, 1999Go). Molecular weights of 52 kDa and 150 kDa were used to convert nanograms to molar concentrations of GST-Rab4/5 and antibody, respectively.

Immunofluorescence localization of Rab4 in cultured rat hepatocytes
Isolated rat hepatocytes were prepared by collagenase digestion and cultured onto glass coverslips coated with 1 mg/ml calf skin collagen as we have described previously (Novikoff et al., 1996Go). After overnight culture, cells were washed in Waymouth's 752/1 medium (Life Technologies) and incubated at 37°C for 60 minutes prior to incubation in Texas-Red–ASOR (5 µg/ml in Waymouth medium) for 20 minutes followed by plasma membrane lysis in 100 units/ml Streptolysin O (Sigma catalogue # S5265) in MEPS (35 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 5 mM EGTA, 5 mM MgCl2, 0.2 M sucrose, pH 7.1), 4 mM DTT, protease inhibitor cocktail (Sigma cat. #p8340 used 1:50 dilution) for 5 minutes. Cells were then blocked in PMEE + 2 mg/ml BSA + 5 mg/ml casein for 5 minutes, incubated in anti-Rab4 monoclonal antibody (30 nM) for 30 minutes, washed, incubated in Cy-2 anti-mouse IgG (15 mg/ml) for 30 minutes, washed, mounted for microscopy in 50% glycerol/PMEE and photographed immediately. Cells lacking Texas-Red–ASOR and primary antibody were completely black under these conditions. Rab4 immunofluorescence of fixed cells was unsuccessful as paraformaldehyde fixative reduced Rab4 staining. Images were captured on a Biorad Radiance 2000 confocal microscope at the analytical imaging facility of the Albert Einstein College of Medicine.

Statistical analysis
Statistical analysis was performed using Chi-square or Student's t-tests as appropriate (SigmaStat v 2.0, SPSS, Chicago, IL).


    Results
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 Materials and Methods
 Results
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 References
 
Identification of proteins associated with ligand-containing early endocytic vesicles
Colocalization of proteins with fluorescent MT bound, ligand (ASOR)-containing early endocytic vesicles was examined by immunofluorescence microscopy (Murray et al., 2002Go) using a series of specific antibodies (Table 1). As we have previously shown, these early endocytic vesicles have substantial association with the asialoglycoprotein receptor (ASGPR) and kinesins, but little association with dynein or dynactin (Bananis et al., 2000Go). We find that approximately 65% of ASOR-containing vesicles are associated with the minus-end kinesin, KIFC2, consistent with our findings of endocytic vesicle minus-end movement in the absence of dynein (Bananis et al., 2000Go). Additional results regarding KIFC2 are presented below.


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Table 1. Identification of proteins associated with MT-bound ASOR-containing endocytic vesicles

 

Approximately 23% of vesicles colocalized with dynamin II, a `pinchase' that is thought to mediate budding of clathrin-coated vesicles from plasma membrane and Golgi (McNiven et al., 2000Go). Although Rab7, clathrin, or Rab5 antibodies were found localized to MT-attached vesicles, few of these vesicles contained Texas-Red–ASOR (Table 1, Fig. 1D-F). In contrast, Rab4 immunolocalized to approximately 80% of MT-bound ASOR-containing vesicles (Table 1, Fig. 1A-C). There was no association of these vesicles with Rab-GDI, and we were unable to find this protein in the vesicle preparation by immunoblot (data not shown). That Rab4 co-localized with ASOR-containing endocytic vesicles in vivo was demonstrated in overnight cultured rat hepatocytes that were incubated for 20 minutes at 37°C with Texas-Red–ASOR. As seen in Fig. 2, there was frequent association of Rab4 with Texas-Red–ASOR-containing vesicles. We were unable to demonstrate association of these vesicles with Rab5.



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Fig. 1. Colocalization of early endocytic vesicles with Rab4 but not with Rab5. Representative studies are shown in which Texas-Red–ASOR and rhodamine-labeled MTs are visualized in panels A and D. Rab4 is immunolocalized in panel B, while Rab5 is immunolocalized in panel E. Merged images (C,F) reveal that the majority of MT-bound ligand-containing vesicles are associated with Rab4 (C). There is little colocalization of MT-bound ASOR-containing vesicles with Rab5 (F), although Rab5 is associated with many vesicles that do not contain ASOR.

 


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Fig. 2. Immunofluorescence localization of Rab4 in cultured rat hepatocytes. Overnight cultured rat hepatocytes grown on collagen-coated glass coverslips were incubated with Texas-Red-labeled ASOR for 20 minutes, treated with streptolysin O to permeabilize the plasma membrane, and stained with anti-Rab4 monoclonal antibody and Cy2-labeled anti-mouse antibody. Merged images of confocal sections and corresponding phase images of two representative cells are presented, revealing punctate areas of overlap (arrows) between endocytosed ASOR (red) and Rab4 (green). Bar, 10 µm.

 

Antibody affinities for Rab4 and Rab5
Differences in vesicle association of antibodies against Rabs 4 and 5 (Fig. 1, 30 nM antibody) could be due to differential affinities of these antibodies for their respective ligands. Several studies were conducted to examine this possibility. As seen in Fig. 3, colocalization of these antibodies with early endocytic vesicles did not change over a 10-fold range of monoclonal antibody concentration (15-150 nM). In addition, affinities of these antibodies for their respective ligands were determined directly by surface plasmon resonance (SPR). Cuvettes were coated with GST-Rab4 or GST-Rab5 fusion proteins and probed with the corresponding antibody. As seen in Fig. 4A,B, each antibody interacted specifically with the protein to which it was raised. There was no interaction of anti-Rab4 with GST-Rab5, anti-Rab5 with GST-Rab4, or either protein with a connexin 43 C-terminal peptide. Analysis of these data (Fig. 4C) revealed high affinity binding (Kd of 2 nM and 22 nM for Rab4 and Rab5, respectively). Although there was a 10-fold difference in the affinities of these antibodies for their respective Rab's, the results in Fig. 3 indicate that experiments were performed under conditions where this made no important difference in the amount of colocalization that was found. Rab5 was therefore used in our experiments as a control for the actions of Rab4.



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Fig. 3. Colocalization of MT-bound early endocytic vesicles with increasing concentrations of antibodies against Rab4 and Rab5. Monoclonal antibodies against Rab4 or Rab5 at increasing concentrations were perfused into a chamber containing MT-bound Texas-Red–ASOR-labeled vesicles, incubated for 6 minutes and visualized after addition of Cy2-labeled secondary antibody. The plot indicates percent colocalization between MT-bound Texas-Red–ASOR vesicles with Rab4 and Rab5 antibody at the indicated concentrations.

 


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Fig. 4. The determination of affinities of anti-Rab4 and anti-Rab5 to GST-Rab fusion proteins by surface plasmon resonance. Surface plasmon resonance technology was used to measure the binding of anti-Rab4 monoclonal antibody to a cuvette coated with GST-Rab4 (A), and the binding of anti-Rab5 monoclonal antibody to a GST-Rab5-coated cuvette (B). Antibody was added at the indicated concentrations, binding was monitored for 10 minutes, cuvettes were acid washed to remove antibody, and antibody was re-added. An equilibrium plot of final signal versus antibody concentration (C) was used to fit a curve for the apparent affinities of the antibodies, yielding Kd values of 2 and 22 nM for anti-Rab4 and anti-Rab5 monoclonal antibodies, respectively. (D) Endocytic vesicles (total protein ~60 µg) were incubated with 0.5 µM GST-Rab4 for 15 minutes at 37°C. Following extensive washing vesicles were immunoblotted with monoclonal antibody against Rab4.

 

Interaction of recombinant Rab4 and Rab5 with early endocytic vesicles
Rab4 GST fusion protein was purified from transformed E. coli. Initially, binding of GST-Rab4 to endocytic vesicles was examined (Fig. 4D). Endocytic vesicles were incubated with 0.5 µM GST-Rab4 for 15 minutes at 37°C and after multiple washes vesicle pellets were analyzed by immunoblot for GST-Rab4 and endogenous Rab4. As seen in Fig. 4D, the amount of GST-Rab4 bound is equivalent to the endogenous levels on endocytic vesicles. GST-Rab fusion proteins also bound the nucleotides [3H-GDP] and GTP-{gamma}-[35S] as confirmed by filtration assays (data not shown). Additionally, the concentration of Rab4 in liver cells was determined to be 30 nM based upon GST-Rab4 standards and a total cell protein concentration of 65 mg/ml (Hiller and Weber, 1978Go).

Interaction of endosome-bound endogenous Rab4 with Rab-GDI
Rabs are known to be prenylated in vivo and require Rab-GDI to dissociate from membranes (Dirac-Svejstrup et al., 1994Go; Pfeffer et al., 1995Go). As seen by western blot in Fig. 5A, when endocytic vesicles were centrifuged at high speed, Rab4 partitioned into the vesicle pellet following treatment with buffer alone, GDP, or GTP-{gamma}-S, indicating that Rab4 was bound to the vesicles. However, Rab4 appeared in vesicle supernatants after incubation with Rab-GDI (Fig. 5B). Treatment of vesicles with GDP prior to Rab-GDI addition increased the amount of Rab4 in the supernatants (40% by gel densitometry) and concomitantly decreased the amount in the pellet. Treatment with GTP-{gamma}-S prior to Rab-GDI addition reduced Rab4 in the supernatant by 86% and increased the amount in the pellet. The data demonstrate that GDP and GTP-{gamma}-S exchange into Rab4 present on the vesicle under these experimental conditions. Unexpectedly, as seen in Fig. 5C, we found that monoclonal antibody to Rab4 also released Rab4 into the supernatants, and that the amount of release was also increased following GDP treatment. Vesicles were incubated with Rab4 monoclonal antibody, centrifuged and immunoblots were performed on the supernatant and pellets as in the GDI experiments. Rab4 was visible as a faint band running just beneath the 25 kDa band of the monoclonal antibody IgG. Preincubation of the vesicles with GDP lead to an increase in the amount of Rab4 in the supernatants and a corresponding decrease in the amount of Rab4 in the pellets. Similar studies could not be performed with GTP-{gamma}-S, as GTP-{gamma}-S treatment caused reduction of the Rab4 mAb light chain, yielding a large, obscuring band at the 25 kDa position (data not shown).



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Fig. 5. Direct demonstration that Rab-GDI or monoclonal antibody to Rab4 release Rab4-GDP from early endocytic vesicles. Endocytic vesicles were preincubated with buffer or buffer plus 4 mM GDP or 4 mM GTP-{gamma}-S for 15 minutes at 37°C and subsequently incubated in the absence (A) or presence of Rab-GDI (B) or Rab4 monoclonal antibody (Rab4 mAb, C). After centrifugation, Rab4 was detected in supernatants or pellets by western blot as indicated. Rab-GDI caused Rab4 to appear in the supernatants and the amount of Rab4 was increased by preincubation of the vesicles with GDP and decreased by preincubation of the vesicles with GTP-{gamma}-S. Similarly, Rab4 mAb caused Rab4 to appear in the supernatants and the amount was increased by preincubation of the vesicles with GDP. The IgG band at 25 kDa results from crossreactivity of the anti-mouse secondary antibody with the Rab4 mAb that was incubated with the vesicles in the experiment.

 

Release of Rab4-GDP from endocytic vesicles via the anti-Rab4 mAb was also seen by immunofluorescence. Pre-treatment of vesicles with GDP or GDP-{gamma}-S decreased the number of vesicles that stained positive for Rab4 (Fig. 6A-C, Table 2), as compared to untreated controls (Fig. 1A-C, Table 2). Pre-treatment of vesicles with GTP-{gamma}-S did not reduce Rab4 immunofluorescence (Table 2). Dot-blot analysis confirmed that the antibody recognized both GDP and GTP-{gamma}-S forms of Rab4 equally well (data not shown). To confirm that GDP by itself was not responsible for loss of immunofluorescence, we pre-incubated vesicles with GDP, then added 4 mM GTP-{gamma}-S, and finally immunostained for Rab4. This scenario gave bright Rab4 fluorescence similar to control Rab4 staining (Fig. 6D-F, Table 2). No change in dynamin II, ASGPR, or kinesin staining was observed upon pre-incubation with GDP (data not shown).



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Fig. 6. The effect of guanine nucleotides on Rab4 immunolocalization on MT-bound endocytic vesicles. MT-bound Texas-Red–ASOR-containing early endocytic vesicles were incubated for 5 minutes in a motility chamber with buffer containing 4 mM GDP (top panels), or 4 mM GDP followed by an additional 5 minute incubation with 4 mM GTP-{gamma}-S (bottom panels). Monoclonal antibody against Rab4 was perfused into the chamber, incubated for 6 minutes, and visualized after addition of Cy2-labeled secondary antibody. Representative studies are shown in which Texas-Red–ASOR and rhodamine-labeled MTs are visualized in panels A and D. Rab 4 is immunolocalized in panels B and E. Merged images reveal that Rab4 is no longer immunolocalized to vesicles when pre-incubated with GDP (C). However, when GTP-{gamma}-S is perfused after GDP, Rab4 remains associated with these vesicles (F). These results are consistent with removal of Rab4-GDP but not Rab4-GTP from vesicles by the monoclonal antibody.

 

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Table 2. Effect of guanosine nucleotides on Rab4 association with endocytic vesicles

 

Effect of nucleotide treatment on motility and fission of endocytic vesicles
Upon pre-treatment with 4 mM GDP, the number of motile vesicles and the number of fission events in preparations of ASOR-containing MT-bound endosomes appeared to be substantially increased. Fig. 7 shows examples of two microscope fields of endocytic vesicles bound to microtubules, the lower of which has been pretreated with GDP. The small arrows point to examples of moving vesicles while the large arrows show the original vesicle position. Arrow heads mark vesicle fission events, and these are only seen in the GDP case (lower panels). Quantification of over 1600 MT-bound endocytic vesicles in control conditions showed that 22% became motile and of these, 12% underwent fission, in agreement with previous results (Bananis et al., 2000Go). Pre-incubation with GDP or its non-hydrolysable derivative GDP-ß-S, resulted in significantly increased numbers of motile vesicles (32% and 34% respectively, P<0.001). The proportion of motile vesicles undergoing fission was also increased significantly following exposure of vesicles to GDP or GDP-ß-S (22% and 21% respectively, P<0.001), so that any moving vesicle was almost twice as likely to undergo fission after GDP addition. Pre-treatment of vesicles with GTP-{gamma}-S, resulted in a significant decrease of motile vesicles as compared to control (15% versus 22%, respectively, P<0.001). However, the percentage of motile vesicles undergoing fission following GTP-{gamma}-S treatment did not differ from control (P>0.50).



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Fig. 7. Pre-incubation of MT-bound vesicles with GDP results in increased motility and fission. Fluorescent early endocytic vesicles were prepared after injection of a rat with Texas-Red-labeled ASOR. Endocytic vesicles were flowed into a 3 µl microscopy chamber in which Taxol-stabilized rhodamine-labeled MTs had been attached to the glass surface. MT-bound vesicles were incubated for 5 minutes with buffer alone (A) or with buffer containing 4 mM GDP (B). Time in seconds after addition of 50 µM ATP is shown at the bottom of each panel. Arrows point to examples of vesicles bound to MTs. In subsequent panels, smaller arrows indicate motile vesicles while larger arrows point to the original vesicle position (A,B). In B, arrowheads point to vesicles undergoing fission. Bar, 10 µm.

 

Vesicles were also treated with GDP followed by Rab-GDI. These conditions released Rab4 from the vesicles (Fig. 5, Table 2). However, the number of motile vesicles and fission events were not substantially different from GDP treatment without Rab-GDI (Fig. 8). We did observe that microtubule `bending' or `gliding' was reduced by Rab-GDI. The microtubules are attached to glass via pre-coating the glass with DEAE-dextran; vesicles are then incubated with the microtubules. Upon ATP addition, some of the microtubules bend or glide small distances across the glass due to motors present in the vesicle preparation. This activity appeared reduced after Rab-GDI treatment. However, as stated above, the number of moving and fissioning ASOR-containing vesicles was not changed.



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Fig. 8. Effects of guanosine nucleotide analogues on MT-based motility and fission of early endocytic vesicles. MT-bound vesicles were incubated for 5 minutes with buffer alone (control) or with buffer containing 4 mM GTP-{gamma}-S, GDP-{gamma}-S, GDP, GDP plus 0.5 µM Rab-GDI, or 2 µM GST-Rab4 as indicated. Guanine nucleotides were exchanged into GST-Rab4 by preincubation immediately before addition to vesicles. ATP (50 µM) was added to produce vesicle motility, and this activity was scored. The bars in panel A indicate the percentage of MT-bound vesicles that moved upon ATP addition. For each experiment, the number of motile vesicles versus the total number of MT-bound vesicles that were examined is in parentheses. The bars in panel B indicate the percentage of moving vesicles that underwent fission. For each experiment, the number of vesicles that underwent fission versus the number of motile vesicles is in parentheses. *P<0.001 versus control.

 

Rab4-GTP-{gamma}-S and Rab4–GDP were assayed for their effect on motility. GST-Rab4 was incubated with either GDP or GTP-{gamma}-S, resulting in nucleotide incorporation, and these were then incubated with microtubule bound vesicles, washed, and motility and fission were scored. GST-Rab4-GTP-{gamma}-S substantially reduced motility and fission compared to treatment with GTP-{gamma}-S alone (percent moving: 5% versus 15%, fission rate: 4% versus 12%), whereas GST-Rab4-GDP was similar to GDP treatment alone (percent moving: 29% versus 32%, fission rate: 17% versus 22%). These data indicate that the GTP-{gamma}-S form of Rab4 is inhibitory toward motility.

GDP enhances minus-end-directed movement of early endocytic vesicles
The direction of movement of endocytic vesicles bound to polarity marked microtubules with or without pretreatment with 4 mM GDP was also determined. As seen in Fig. 9, following pre-incubation with buffer alone, 52% of motile MT-bound early endocytic vesicles moved towards the plus end while 48% moved towards the minus end. These results are consistent with our previous observations (Bananis et al., 2000Go; Murray et al., 2000Go). In contrast, following pre-incubation with buffer containing 4 mM GDP, only 33% of motile MT-bound early endocytic vesicles moved towards the plus end of polarity marked microtubules while 67% moved towards the minus end (P<0.01).



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Fig. 9. GDP enhances minus-end-directed motility of early endocytic vesicles. Fluorescent-polarity-marked microtubules were prepared as described (Murray et al., 2000Go). MT-bound vesicles were incubated for 5 minutes with buffer alone or with buffer containing 4 mM GDP, following which 50 µM ATP was added to produce vesicle motility. The bars indicate the percentage of vesicles that moved upon ATP addition in the minus-end or plus-end directions. For each experiment, the number of motile vesicles in either direction versus the total number of motile vesicles that were examined is in parentheses. *P<0.01.

 

Antibody to Rab4 inhibits motility of early endocytic vesicles
Studies were also performed to examine the effect of antibodies to Rab4 and Rab5 on vesicle motility and fission (Table 3). Using similar methodology, we previously showed that antibodies towards kinesin, but not dynein, inhibited early endocytic vesicle motility and fission (Bananis et al., 2000Go). This was also demonstrated for endocytic vesicles derived from HeLa cells (Nielsen et al., 1999Go). In the current experiments, endocytic vesicles were bound to MTs, incubated for 6 minutes with 30 nM anti-Rab4 or anti-Rab5 IgG, washed, and motility and fission of ASOR-containing vesicles were quantified upon addition of 50 µM ATP. The Rab5 antibody did not alter motility. In contrast, motility was markedly reduced by anti-Rab4 antibody. Addition of fluorescent secondary antibody prior to ATP addition revealed that the small number of ASOR-containing vesicles that moved in the presence of Rab4 antibody did not stain for Rab4 (data not shown). Neither antibody reduced the rate of vesicle fission. There were no changes in motility or fission of vesicles with addition of anti-dynamin II IgG (data not shown) or anti-ASGPR IgG (Bananis et al., 2000Go).


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Table 3. Motility and fission of endocytic vesicles in the presence of Rab antibodies

 

To determine whether guanine nucleotides alter the effect of anti-Rab4 on motility, vesicles were pre-incubated with GTP-{gamma}-S, GDP, or GDP-{gamma}-S, followed by anti-Rab4 and ATP. GTP-{gamma}-S followed by anti-Rab4 produced no further inhibition of motility than anti-Rab4 alone. However, GDP or GDP-{gamma}-S pre-incubation released the inhibition of the Rab4 antibody (Table 3). Under this condition, motility was slightly lower than GDP treatment without anti Rab4 antibody. However, fission activity increased to the high levels seen with GDP (Table 3, Fig. 8). These results suggest that antibody interaction with GTP-Rab4 is responsible for inhibition of motility. Addition of GDP results in formation of Rab4-GDP, allowing solubilization of Rab4 by anti-Rab4 antibody (Figs 5, 6), and preventing the antibody from inhibiting motility. GTP-{gamma}-S treatment did not induce further inhibition by Rab4 antibody as motility was already suppressed to near completion.

The mechanism by which Rab4 antibody inhibits motility is not yet known, but we favor the interpretation that crosslinking of vesicle-associated endogenous Rab4-GTP by antibody potentiates its inhibitory effect on motility. Inhibition of microtubule-based motility by specific antibodies has been used by many investigators (e.g. McDonald et al., 2002Go; Nielsen et al., 1999Go), and blockage of enzymatic activity (ATP hydrolysis) does not appear necessary for this effect. Instead steric interference or structural crosslinking has been offered as an explanation (Brady et al., 1990Go).

Functional association of KIFC2 on early endocytic vesicles
Our previous studies demonstrated that ASOR-containing early endosomes did not show immunofluorescence for cytoplasmic dynein, nor did they respond to dynein inhibitors (antibody and vanadate), yet these endosomes moved bi-directionally on microtubules (Bananis et al., 2000Go). Fig. 9 demonstrates that GDP increases minus-end movement of the endosomes. We therefore predicted that minus-end-directed kinesins are present on the endosomes and that these kinesins are somehow stimulated by pre-incubation with GDP. Three minus-end kinesins are known to exist in mammals, the C-terminal motor domain proteins, KIFC1, 2, and 3 (Miki et al., 2001Go). KIFC2 and KIFC3 are known to be associated with intracellular organelles (Hanlon et al., 1997Go; Noda et al., 2001Go), and a commercial available antibody to KIFC2 was available to probe our vesicle fractions for this minus-end-directed kinesin.

Because KIFC2 has been described as neural specific and was not detected by northern and western blot in liver (Saito et al., 1997Go), this issue was reinvestigated using immunoblot analysis to liver subcellular fractions. As seen in Fig. 10A and in agreement with previous studies (Saito et al., 1997Go), KIFC2 is not detected in liver homogenate. However, KIFC2 is detected in the final endocytic vesicle fraction when assayed at same total protein concentration. This fraction is heavily purified from total cell protein and devoid of soluble protein and most liver membrane components (Murray et al., 2000Go). The same antibody was used to confirm the absence of KIFC2 in a mouse knockout strain (Yang et al., 2001Go). We therefore conclude that KIFC2 is weakly expressed in liver (and possibly other tissues) and concentrates in the fraction containing ASOR-containing early endocytic vesicles.



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Fig. 10. KIFC2 is highly enriched and functional on endocytic vesicles. (A) Fifteen micrograms of total protein from rat liver homogenate, PNS, cytosol, S-200 fraction and purified endocytic vesicles were subjected to 10-20% gradient SDS-Page and immunoblotted with antibody to KIFC2, as described in Materials and Methods. (B,C) MT-bound endocytic vesicles were incubated for 6 minutes with buffer alone or buffer containing affinity purified anti-KIFC2, following which 50 µM ATP was added to produce vesicle motility. In some studies, fluorescent polarity marked microtubules were used (C). The bars in panel B indicate the percentage of MT-bound vesicles that moved upon ATP addition. The number of motile vesicles versus the total number of MT-bound vesicles that were examined is in parentheses. The bars in panel C indicate the percentage of vesicles that moved upon ATP addition in the minus-end or plus-end directions. For each experiment, the number of motile vesicles in either direction versus the total number of motile vesicles that were examined is in parentheses. *P<0.002 versus control.

 

The effect of the KIFC2 antibody on motility and direction of movement of early endocytic vesicles on MTs was also examined. In the presence of KIFC2 antibody, overall motility of vesicles was inhibited by over 60% as compared to control (Fig. 10B), due largely to a decrease in minus-end-directed motility (19% vs. 47%, P<0.002, Fig. 10C).


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we examined specific nucleotides and Rab GTPases as potential regulators of endosomal processing, using endocytosis mediated by the hepatocyte receptor for desialylated glycoproteins as our model. The biochemical characteristics of this receptor system make it particularly advantageous for elucidation of subcellular processing events (Oda et al., 1995Go; Samuelson et al., 1988Go; Wolkoff et al., 1984Go). To identify early endocytic vesicle-associated proteins that might regulate processing, we used fluorescence microscopy procedures performed in a miniaturized system that permits real-time evaluation of MT-associated endocytic vesicles without fixation (Murray et al., 2002Go; Murray et al., 2000Go; Nielsen et al., 2001Go). As summarized in Table 1, MT-bound early endocytic vesicles were screened for the presence of specific candidate proteins. These studies identified Rab4 as a protein that is highly associated with these vesicles under control conditions. Endogenous Rab4 has been localized to early endosomes and small vesicles in PC12 cells by electron microscopy (de Wit et al., 2001Go), and transfected Rab4 has been colocalized to endocytic vesicles in MDCK cells, and to specific endocytic domains in A431 cells (Sonnichsen et al., 2000Go). We show that endogenous Rab4 co-localizes with endocytic vesicles following uptake of fluorescent ASOR by primary-cultured rat hepatocytes (Fig. 2). Therefore colocalization of Rab4 with purified endocytic vesicles is presumed to be a reflection of in vivo events.

The Rab family of small GTPases is composed of a large number of similar but biologically distinct proteins. They have been described as functioning as molecular switches that oscillate between GTP- and GDP-bound forms (Zerial and McBride, 2001Go). In the GTP, or activated forms, Rabs associate with membranes and form a scaffold with specific cytosolic proteins (Nagelkerken et al., 2000Go; Segev, 2001Go; Zerial and McBride, 2001Go). Although the best established function of Rabs is in promoting membrane fusion (Rodman and Wandinger-Ness, 2000Go; Takai et al., 2001Go; Zerial and McBride, 2001Go), additional functions of specific Rabs have recently been elucidated (Segev, 2001Go; Takai et al., 2001Go; Zerial and McBride, 2001Go). These include Rab6 serving as an adaptor for a Golgi-associated kinesin-like protein termed Rabkinesin-6 (KIF20A) (Echard et al., 1998Go). Rabkinesin-6 associates specifically with the GTP form of Rab6, serving as a putative adaptor complex to link Golgi membranes with MTs (Echard et al., 1998Go). Other studies have shown binding of myosin Va to Rab27a (Hume et al., 2001Go; Strom et al., 2002Go). It has been suggested that Rab27a is necessary for the recruitment of myosin Va to melanosomes, resulting in their distribution at the periphery of melanocytes (Hume et al., 2001Go; Strom et al., 2002Go). It was also found that Rab4 can bind to the light intermediate chain-1 of cytoplasmic dynein (Bielli et al., 2001Go).

Other studies indicated that populations of early endocytic vesicles are associated with Rab4 and Rab5 (Chavrier et al., 1997Go; Di Fiore and de Camilli, 2001Go; Nielsen et al., 1999Go; Sonnichsen et al., 2000Go; Trischler et al., 1999Go) and it has been suggested that Rab5 regulates motility of transferrin-containing early endocytic vesicles on MTs (Nielsen et al., 1999Go). In this system, in vitro bi-directional motility of endosomes is also mediated by kinesins rather than dynein. However these investigators found that treatment with Rab-GDI abolishes motility and that treatment with Rab-GDI-Rab5, resulting in Rab5 delivery, increases motility. In our system Rab-GDI has minimal effect on the motility of ASOR vesicles (Fig. 8). However we do see a decrease in microtubule gliding, indicating that the activity of motors associated with other vesicles in the preparation may be decreased. Furthermore, we find that Rab5 is associated with only 9% of the ASOR-containing vesicles (Table 1, Fig. 1). Among the differences between the two systems, the earlier study (Nielsen et al., 1999Go) used transferrin vesicles from cultured HeLa cells whereas the ASOR vesicles are isolated from rat liver, and the ASOR vesicles do not require cytosol for motility.

Under normal conditions, transferrin does not dissociate from its receptor, and recycles shortly after internalization. It is possible that early endocytic vesicles from a receptor-ligand system (e.g. ASGPR/ASOR) in which ligand dissociates from receptor and targets to lysosomes requires distinct regulators. It must also be considered that the ASOR vesicles have already passed through a Rab5 compartment (Sonnichsen et al., 2000Go). Similarly, other proteins such as Rab7, EEA1, clathrin, or dynactin that are known to interact with the endosome at different processing steps are found only as minor fractions on the ASOR vesicles. Although previous studies of Rab4 in endocytic processing suggested a role in formation of recycling endocytic vesicles (Chavrier et al., 1997Go; Daro et al., 1996Go; McCaffrey et al., 2001Go; Nagelkerken et al., 2000Go; Rodman and Wandinger-Ness, 2000Go; van der Sluijs et al., 1992Go), evidence has also been provided for two populations of early endosomes, with Rab4 being enriched in one of these populations that was distinct from the transferrin receptor (Sheff et al., 1999Go), and a role for Rab4 in fission of vesicles from a Rab5-mediated fusion compartment has also been reported (Chavrier et al., 1997Go).

Our studies provide several lines of evidence that Rab4 is a regulator of early endocytic vesicle processing. These include inhibition of vesicle motility by addition of recombinant Rab4-GTP-{gamma}-S. Similar to our findings, others have found that specific Rab proteins in their GTP-bound form inhibit specific vesicle transport pathways. A mutant Rab2 in its GTP-bound form blocked ER-to Golgi transport of VSV-G (Tisdale, 1999Go). Rab3A in its GTP-bound form is a negative modulator of cholecystokinin secretion from STC-1 cells (Gevrey et al., 2001Go). Overexpression of Rab7 specifically inhibited late endocytic vesicle motility in HeLa cells (Lebrand et al., 2002Go).

Addition of 4 mM GDP to the endocytic vesicles converts Rab4 into its GDP state (Fig. 5) and enhances minus-end-directed motility (Fig. 9) and fission (Fig. 8). Our data suggest that conversion of Rab4-GTP to Rab4-GDP is sufficient for this effect, as results are identical whether or not Rab4-GDP is removed from vesicles by Rab-GDI. In the absence of added GDP, both plus- and minus-end-directed motility are equally active. Following GDP addition, plus-end-directed motility is unaffected but minus-end-directed motility is doubled (Table 4).


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Table 4. Calculated effect of GDP on directional motility of ASOR-containing early endocytic vesicles

 

Because dynein is neither present nor functional on the vesicles (Bananis et al., 2000Go) we propose that the GDP pretreatment leads to activation of minus-end-directed kinesins that are then responsible for the increased minus-end movement and increased fission rate. Consistent with this suggestion, we have shown that a minus-end-directed kinesin, KIFC2, is present on these vesicles and that antibody against KIFC2 inhibits minus-end motility (Fig. 10).

Our hypothesis for how sorting of the asialoglycoprotein receptor and its ligand is regulated within endocytic vesicles is shown in Fig. 11. Ligand (ASOR) binds to its receptor (ASGPR) at the cell surface where it is internalized into an endocytic vesicle. The vesicle binds to microtubules through plus- and minus-end-directed kinesins. GTP-Rab4 is present on the vesicle and inhibits minus-end kinesins (possibly KIFC2). Hydrolysis of GTP on Rab4 releases the minus-end kinesin inhibition, allowing minus-end-directed movement, resulting in fission and sorting of the vesicle contents along the microtubule.



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Fig. 11. Model of the proposed mechanism for regulation by Rab4 of early endocytic vesicle sorting of the asialoglycoprotein receptor and ligand. The shaded area represents the cell exterior and the vesicle interior, the red tube represents a microtubule with its plus (+) and minus (-) ends specified, the arrow represents passage of time on the scale of tens of seconds. Ligand (ASOR) binds to its receptor (ASGPR) at the cell surface, where it is internalized into an endocytic vesicle. The vesicle binds to microtubules through plus- and minus-end-directed kinesins. GTP-Rab4 present on the vesicle inhibits minus-end kinesins (KIFC2). Hydrolysis of GTP on Rab4 releases this inhibition, allowing minus-end-directed movement of the vesicle, resulting in fission and sorting of its contents along the microtubule. For simplicity Rab4 is shown binding directly to the minus-end kinesin, and only the minus-end motor is shown moving. Endogenous Rab-GDI could remove Rab4-GDP from the vesicle, allowing Rab4 to be recycled. In cultured rat hepatocytes, ligand is eventually (after 30-60 minutes) sorted to lysosomes (Wolkoff et al., 1984Go), which reside near the cell center at the minus ends of microtubules. However, early endocytic sorting events do not demonstrate simple progression toward the cell center and instead show multiple plus- and minus-end-directed movements (Murray et al., 2000Go).

 

We have previously demonstrated sorting of receptor and ligand by vesicle fission events along microtubules, and we proposed that multiple rounds of fission could yield segregated receptor and ligand (Bananis et al., 2000Go). In the current report we show: (1) Rab4 is associated with the vesicles; (2) purified GTP-{gamma}-S-Rab4 but not GDP-Rab4 inhibits vesicle motility; (3) GDP exchanges into endogenous Rab4; and (4) GDP addition results in increased minus-end-directed motility. Furthermore we show that the minus-end-directed kinesin, KIFC2 is present on the vesicles.

We propose that the addition of GDP in vitro mimics the endogenous event of GTP hydrolysis on Rab4, both yielding GDP-Rab4. In the cell, endogenous Rab-GDI could remove Rab4-GDP from the vesicle, allowing Rab4 to be recycled. Currently we are investigating whether Rab4-GTP interacts directly (Bielli et al., 2001Go; Echard et al., 1998Go; Hume et al., 2001Go; Strom et al., 2002Go) or through a protein scaffold (Setou et al., 2000Go; Verhey et al., 2001Go) with KIFC2. Identification of Rab4-interacting proteins on early endocytic vesicles will provide an important key to unraveling further the complexities of endosome trafficking, segregation and processing.


    Acknowledgments
 
We thank Ira Mellman for kindly providing the Rab4 and Rab5 cDNAs in pBlueScriptKS+, and to Pijun Wang for help in preparation of the Rab-GST fusion proteins. We are also grateful for the help and support provided by Heather Duffy and David Spray in performing the surface plasmon resonance studies. This work was supported by National Institutes of Health grants DK41918 and DK41296. E.B. is presently supported by NCI Training Grant CA09475. He was the recipient of a student research award and J.W.M. was the recipient of a fellow research award, both from the American Liver Foundation.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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