Membrane Targeting of a Rab GTPase That Fails to Associate with Rab Escort Protein (REP) or Guanine Nucleotide Dissociation Inhibitor (GDI)*

Jean H. OvermeyerDagger , Amy L. Wilson§, and William A. MalteseDagger

From the Dagger  Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804 and § Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, February 16, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The targeting of various Rab proteins to different subcellular compartments appears to be determined by variable amino acid sequences located upstream from geranylgeranylated cysteine residues in the C-terminal tail. All nascent Rab proteins are prenylated by geranylgeranyltransferase II, which recognizes the Rab substrate only when it is bound to Rab escort protein (REP). After prenylation, REP remains associated with the modified Rab until it is delivered to the appropriate subcellular membrane. It remains unclear whether docking of the Rab with the correct membrane is solely a function of features contained within the prenylated Rab itself (with REP serving as a "passive" carrier) or whether REP actively participates in the targeting process. To address this issue, we took advantage of a mutation in the alpha 2 helix of Rab1B (i.e. Y78D) that abolishes REP and GDI interaction without disrupting nucleotide binding or hydrolysis. These studies demonstrate that replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL motif permits this protein to be prenylated by geranylgeranyltransferase I but not II both in cell-free enzyme assays and in transfected cells. Subcellular fractionation and immunofluorescence studies reveal that the prenylated Rab1B(Y78D)CLLL, which remains deficient in REP and GDI association is, nonetheless, delivered to the Golgi and endoplasmic reticulum (ER) membranes. When the dominant-negative S22N mutation was inserted into Rab1B-CLLL, the resulting monoprenylated construct suppressed ER right-arrow Golgi protein transport. However, when the Y78D mutation was added to the latter construct, its inhibitory effect on protein trafficking was lost despite the fact that it was localized to the ER/Golgi membrane. Therefore, protein interactions mediated by the alpha 2 helical domain of Rab1B(S22N) appear to be essential for its functional interaction with components of the ER right-arrow Golgi transport machinery.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Rab family of proteins consists of more than 40 Ras-related GTPases, each involved in specific steps of vesicular transport (1-3). Rab1A and Rab1B are two of the most extensively studied members of the Rab family. Both proteins are found in membranes of the ER,1 Golgi apparatus and intermediate vesicles between these compartments (4, 5). As suggested by their localization, these GTPases function in the anterograde trafficking of proteins from the ER to the Golgi compartment (4, 6, 7).

According to accepted models (3, 8, 9), Rab proteins cycle on and off donor and acceptor membranes in a guanine nucleotide-dependent fashion. In the GDP-bound form, which is presumed to represent the inactive state, the Rab proteins are typically associated with an accessory protein, guanine nucleotide dissociation inhibitor (GDI), in the cytosol. Upon delivery to the membrane, interaction with an exchange factor promotes substitution of GTP for GDP, placing the Rab protein in the active conformation (10). The activated Rab protein then facilitates the targeting of a budding transport vesicle to the correct acceptor membrane by mediating vesicle-N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) interactions with the corresponding target-SNARE (t-SNARE) (11, 12). At the acceptor membrane, the intrinsic GTPase activity of the Rab protein may be stimulated by interaction with a GTPase-activating protein, returning it to the GDP-bound conformation. In this form, the Rab protein can be removed from the membrane by GDI, completing the cycle.

All Rab proteins are modified post-translationally by 1 or 2 20-carbon geranylgeranyl groups that are linked covalently to cysteine residues at their C termini. This isoprenoid modification (prenylation) is necessary for two facets of Rab function. First, it is required for association of the GTPase with the membrane (13, 14). Second, it promotes optimal interaction of the GDP-bound form of the Rab protein with GDI (15, 16). Prenylation of Rab proteins is catalyzed by geranylgeranyltransferase type II (GGTase II) (17, 18). GGTase II will prenylate the target cysteine residues only if the nascent Rab substrate is first bound to a carrier protein termed Rab escort protein (REP) (19, 20). REP initially binds to the Rab protein while it is in the GDP-bound state (21). After the prenyl groups are added to the C terminus of the Rab protein, GGTase II is released, whereas REP remains bound to the prenylated protein and delivers it to the membrane. Chavrier et al. (22) used chimeric proteins to show that the hypervariable domain near the C terminus of each Rab protein contains information required for targeting to a specific subcellular membrane compartment. However, the precise role of the chaperone (i.e. REP) in this process has yet to be defined. One possibility is that REP acts cooperatively with the Rab protein to promote association with a specific docking complex at the target membrane. An alternative possibility is that REP is a passive carrier that does not interact directly with putative Rab-docking complexes at the acceptor membrane.

In the present study we explore the role of REP in the membrane targeting of Rab1B, taking advantage of our previous observations that amino acid substitutions within the Rab1B alpha 2-helix (e.g. Tyr-78 right-arrow Asp) completely prevent association with REP (23). By changing the two C-terminal cysteine residues of Rab1B(Y78D) to a CLLL motif, we were able to convert the protein to a substrate for geranylgeranyltransferase type I (GGTase I), which can modify monomeric GTPases in the absence of REP. Despite its inability to associate with REP or GDI, the Rab1B(Y78D)CLLL construct was delivered to ER membranes. However, when a dominant-negative mutation was introduced into the same protein, it failed to suppress ER right-arrow Golgi transport. Thus, although membrane targeting of Rab1B can occur in the absence of REP, it appears that protein interactions mediated either directly by the Rab1B alpha 2 helical domain or indirectly by formation of the REP·Rab1B complex are essential for functional association of Rab1B with the vesicular transport machinery.

    MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
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Generation of Rab1B Constructs-- cDNAs encoding MycRab1B(Delta CC), which lacks the two terminal cysteine residues, MycRab1B(wt) and MycRab1B(Y78D), were generated as described previously (23, 24). Two additional constructs involving an amino acid substitution (S22N) or replacement of the two C-terminal cysteine residues with CLLL were created by polymerase chain reaction modification of the rab1B cDNA using Pfu polymerase (Stratagene, La Jolla, CA) and appropriate oligonucleotide primers. The cDNAs were subcloned into pCMV5 (25) for expression in mammalian cell lines and into pET17b (Novagen, Madison, WI) for expression in Escherichia coli. The sequences of all constructs were confirmed by dye-terminator cycle sequencing using an ABI 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). Several of the constructs were subsequently subcloned into a pCMV5 vector that had been modified by polymerase chain reaction to encode an in-frame HA epitope tag (YPYDVPDYA) instead of the Myc tag at the N terminus of the expressed protein. Vectors encoding T7 epitope-tagged REP (pCMVREP1) and FLAG-tagged GDIalpha (pCMVGDIalpha ) were generated as described previously (23, 26).

Prenylation of Rab Proteins in Vitro-- Expression of recombinant MycRab1B proteins was induced in E. coli BL21(DE3)pLysS, and bacterial lysates were prepared as described previously (26). Total protein was determined by the method of Bradford (27), and the relative amount of each MycRab1B protein in each bacterial lysate was determined by immunoblot analysis using a monoclonal antibody (9E10) against the Myc epitope, as described previously (24). The abilities of different Rab1B constructs to serve as substrates for GGTase II were compared by adding aliquots of bacterial lysate containing equal amounts of Rab protein to a 50-µl reaction mixture consisting of 50 mM HEPES, pH 7.4, 1.0 mM dithiothreitol, 5.0 mM MgCl2, 0.2 mM Nonidet P-40, 1 µCi of [3H]GGPP, 20 ng of recombinant REP, 20 ng of recombinant GGTase II. Prenylation of the bacterially expressed Rab proteins by GGTase I was determined in a 50- µl reaction mixture consisting of 50 mM Tris, pH 7.7, 20 mM KCl, 5.0 mM MgCl2, 25 µM ZnCl2, 1.0 mM dithiothreitol, 0.5 mM Zwittergent 3-14, 1 µCi of [3H]geranylgeranyl pyrophosphate, and 10 ng of purified GGTase I. The reactions were stopped after 90 min at 37 °C by the addition of 1× SDS sample buffer (28). Three quarters of each assay sample was subjected to SDS-PAGE and fluorography to determine incorporation of [3H]geranylgeranyl moiety into the recombinant Rab protein. The remainder of each sample was subjected to immunoblot analysis to confirm equal protein loading in the assays.

Subcellular Fractionation of Rab Proteins Expressed in HEK293 Cells-- Cells expressing various MycRab1B constructs were harvested from 100-mm dishes 24 h after transfection. The cells were collected by centrifugation at 500 × g for 5 min, and the pellet was homogenized in 80 mM PIPES, pH 6.8, 5.0 mM EGTA, 1.0 mM MgCl2, 0.05% Triton X-100, and Complete Mini-EDTA-Free protease inhibitors (Roche Molecular Biochemicals). The resulting cell lysate was centrifuged at 100,000 × g for 30 min at 4 °C in a Beckman TLA100.2 rotor. The supernatant and particulate fractions were analyzed by SDS-PAGE and Western blotting. A rabbit polyclonal antibody against the Myc epitope (Upstate Biotechnology, Inc., Lake Placid, NY) was used to detect the epitope-tagged Rab proteins, with goat anti-rabbit-horseradish peroxidase as the secondary antibody. Quantification of the signal produced by Pierce SuperSignal reagents was performed on a LumiImager (Roche Molecular Biochemicals).

Prenylation of Rab Proteins Expressed in Cultured Cells-- HEK293 cells or NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum in a 5% CO2 atmosphere at 37 °C. Cells were transfected with pCMV vectors containing the indicated Rab constructs using LipofectAMINE PLUS (Life Technologies, Inc.) according to the manufacturer's instructions. One hour after transfection, the cells were changed to medium containing 10 µM lovastatin and 200 µCi of [3H]mevalonolactone (Mev) (2.95 Ci/mmol) with or without 10 µM GGTI-298, a specific inhibitor of GGTase I (provided by S. Sebti, Moffitt Cancer Center, Tampa FL) (29). After 18 h at 37 °C, the cells were washed three times with Hanks' balanced salt solution and disrupted in 200 µl of ice-cold lysis buffer (20 mM HEPES, pH 7.3, 20 mM MgCl2, 150 mM NaCl, 0.75% Nonidet P-40 supplemented with protease inhibitors). All subsequent steps were carried out at 4 °C. Particulate material was removed by centrifugation of the lysates at 10,000 × g for 5 min, and the epitope-tagged Rab proteins were immunoprecipitated by incubation with a mouse monoclonal antibody against the Myc epitope for 2 h. Immune complexes were collected by a 1-h incubation with protein A-Sepharose beads coupled to goat anti-mouse IgG. The beads were washed 3 times with lysis buffer, then the Myc-tagged Rab proteins were eluted in immunoprecipitation sample buffer (50 mM Tris-HCl, pH 6.8, 1.4 mM beta -mercaptoethanol, 2.0% SDS, 30.0% glycerol, 0.025% bromphenol blue). One-tenth of each immunoprecipitate was subjected to immunoblot analysis to compare the recoveries of different Rab constructs, whereas the remainder of the sample was analyzed by SDS-PAGE and fluorography to measure [3H]Mev incorporation.

To determine the subcellular distribution of the prenylated Rab proteins, the metabolic labeling, as described above, was started 3 h after transfection, and cells from three 100-mm dishes were pooled. Cell lysates were centrifuged at 100,000 × g for 30 min at 4 °C to obtain cytosol and membrane-enriched fractions. The Myc-tagged Rab proteins were then immunoprecipitated from each fraction and analyzed as described above.

Carboxymethylation of Rab Proteins Expressed in 293 Cells-- Starting 3 h after transfection, cells were switched to methionine-free Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 40 µCi/ml L-[methyl-3H]methionine (PerkinElmer Life Sciences) and incubated at 37 °C. After 18 h, the cells were harvested and lysed as described in the preceding section. One-tenth of the cell lysate was subjected to SDS-PAGE and immunoblot analysis to confirm MycRab1B expression. The Myc-tagged Rab proteins were immunoprecipitated from the remaining cell lysate as described above. After SDS-PAGE and fluorography, the dried gel was cut into 0.5-cm sections, and the amount of [3H]methanol released by alkaline hydrolysis of protein O-methyl esters in each gel slice was determined as described by Clarke et al. (30).

Assay for Rab Interaction with REP in Cultured Cells-- HEK293 cells were co-transfected with a vector encoding T7-tagged REP-1 (pCMVREP1) and different Myc-tagged Rab1B constructs. After transfection, cells were maintained in medium containing an inhibitor of isoprenoid synthesis (10 µM lovastatin) to prevent prenylation and promote the accumulation of nascent Rab·REP complexes. After 48 h, cells from 2 parallel 100-mm cultures were pooled, and cytosolic fractions were subjected to size-exclusion chromatography on a Superose-12 column, essentially as described by Overmeyer et al. (23). The elution positions of the T7-REP and MycRab1B were determined by immunoblot analysis of the column fractions using antibodies against the epitope tags.

Interaction of Rab Proteins with FLAG-GDIalpha in Intact Cells-- HEK293 cells were co-transfected with a pCMV vector encoding FLAG-tagged GDIalpha and the specified Myc-tagged Rab1B. Twenty-four hours after transfection, cells from a 100-mm culture were harvested, and soluble fractions were prepared as described (26). One-tenth of this fraction was subjected to SDS-PAGE and immunoblot analysis to check for expression of FLAG-GDI and MycRab1B. The remaining sample was subjected to immunoprecipitation with anti-FLAG affinity beads to detect Rab proteins bound to FLAG-GDIalpha , as described in detail by Wilson et al. (26).

Immunofluorescent Localization of Expressed Rab Proteins in 293 Cells-- Cells were seeded in 60-mm dishes containing laminin-coated coverslips and were transfected with the specified vectors. Approximately 24 h later, the coverslips were processed for immunofluorescence microscopy. Cells were fixed and permeabilized with cold methanol for 15 min or fixed with cold 3.0% paraformaldehyde then permeabilized by incubation for 2 min with 0.1% (v/v) Triton X-100 in PBS. Mouse or rabbit antibodies against the Myc epitope were used to detect the Rab1B constructs. Where indicated, the cells were co-incubated with a monoclonal antibody against GM130 (1:25 dilution) (Transduction Laboratories, Lexington, KY), a polyclonal antibody against Rab6 (1:200 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA), or a polyclonal antibody against calreticulin (1:150 dilution) (Affinity Bioreagents, Golden, CO). Rhodamine-conjugated goat anti-mouse IgG (Calbiochem) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma) were used at a 1:100 dilution as the secondary antibodies. Photomicrographs were obtained with a Nikon Eclipse E800 fluorescent microscope equipped with a digital camera. Pseudo-coloring and merging of images were performed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).

Assay for Post-translational Processing of the LDL Receptor-- Cultured 293 cells were co-transfected with pLDLR17, which encodes the human LDL receptor (31), and each of the indicated pCMVRab1B constructs. Twenty-four hours later, cells were pulse-labeled for 30 min with 100 µCi of [35S]Easy-Tag ExpressTM protein-labeling mix (PerkinElmer Life Sciences) in 1.0 ml of methionine-free Dulbecco's modified Eagle's medium (DMEM) followed by a 2-h chase in DMEM plus 10% fetal bovine serum supplemented with 200 µM methionine and 200 µM cysteine. Cells from parallel cultures were harvested immediately after the pulse or after the 2-h chase, and the radiolabeled LDL receptor was immunoprecipitated as described by Castellano et al. (32).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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A REP Binding-deficient Rab1B Mutant Can Be Prenylated after Changing the C-terminal Motif-- Mutations in the predicted alpha 2 helix in the Switch-2 domain of Rab1B (e.g. Tyr- 78 right-arrow Asp) disrupt the ability of the GTPase to interact with REP, without impairing GTP binding or hydrolysis (23). Consequently the same mutations prevent REP-dependent prenylation of Rab proteins by GGTase II (23, 33). We hypothesized that an alpha 2 helix mutant might be used to determine whether or not REP is required for the delivery of Rab1B to intracellular membranes, provided that the GTPase could be prenylated by an alternate REP-independent mechanism. To achieve this goal, we took advantage of our previous finding that Rab8, which ends with a C-terminal CAAL2 prenylation motif similar to that in the Rac and Rho GTPases, can be modified either by the REP-dependent GGTase II or by the REP-independent GGTase I, which does not require the formation of a REP·Rab complex (33). Hence, the two C-terminal cysteine residues of Rab1B(wt) and Rab1B(Y78D) were replaced with CLLL to form a GGTase I recognition motif (34, 35). Cell-free assays were used to compare the prenylation of the various recombinant Rab1B proteins by GGTase I versus GGTase II. As seen in Fig. 1, Rab1B(wt), which ends with a CC motif, is a substrate only for GGTase II. The addition of the CLLL motif to the C terminus of this protein permits it to be prenylated by either GGTase I or GGTase II. Separate studies in which REP was omitted from the reaction mixture showed that prenylation of Rab1B(wt) or Rab1B-CLLL by GGTase II was entirely dependent on the presence of the escort protein (data not shown). On the other hand, the addition of REP to the GGTase I reaction neither permitted prenylation of Rab1B(wt) nor enhanced prenylation of Rab1B-CLLL by GGTase I (data not shown). Most importantly, the studies in Fig. 1 show that by substituting the normal CC motif with CLLL, the REP binding-deficient Rab1B(Y78D) mutant could be prenylated in a REP-independent manner by GGTase I. 


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Fig. 1.   Prenylation of recombinant Myc-Rab1B proteins by GGTase I versus GGTase II. Aliquots of E. coli lysate containing the indicated recombinant Myc-Rab1B proteins were geranylgeranylated in cell-free reactions. The top panel depicts an immunoblot showing the amount of MycRab1B present in 25% of each prenylation reaction. The bottom panel shows the corresponding fluorograph of the remaining 75% of the reaction, demonstrating the [3H]geranylgeranyl pyrophosphate incorporation into the same protein.

To verify that the results of the cell-free assays accurately predicted the ability of the C-terminal CC right-arrow CLLL substitution to permit prenylation of the Rab1B(Y78D) mutant in intact cells, Myc-tagged Rab1B constructs were overexpressed in 3T3 cells, and the relative incorporation of [3H]mevalonate into immunoprecipitated proteins was compared (Fig. 2). Mevalonate is an isoprenoid precursor that is incorporated into the geranylgeranyl pyrophosphate substrate used by either GGTase I or GGTase II. Incorporation of this precursor was measured in the presence and absence of GGTI-298, a specific inhibitor of GGTase I (29). Several general conclusions can be drawn from the results. First, when both GGTase I and GGTase II were active (no inhibitor), Rab1B(Y78D) with the normal CC motif was not labeled by [3H]Mev, whereas the same protein with the added CLLL motif was clearly prenylated. Second, the prenylation of Rab1B(Y78D)CLLL was completely prevented by the inhibitor, GGTI-298. Taken together, these results confirm that the modification of Rab1B(Y78D)CLLL in vivo is catalyzed by GGTase I, as indicated by the cell-free assay (Fig. 1). Surprisingly, the wild-type Rab1B-CLLL, which has only a single cysteine available for modification, showed a greater incorporation of [3H]Mev per unit protein than the wild-type Rab1B with the original CC motif. However, it is important to bear in mind that this type of study does not provide a stoichiometric analysis of prenylation, because overexpression results in the accumulation of a large pool of non-prenylated Rab1B in the transfected cells (26). Thus, the increased incorporation of [3H]Mev into Rab1B-CLLL, compared with Rab1B, may reflect the fact that two different GGTase enzyme systems are able to prenylate the Rab1B-CLLL, whereas only the REP/GGTase II system can prenylate Rab1B with the CC ending.


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Fig. 2.   Prenylation of Myc-Rab1B proteins expressed in cells in the presence or absence of a GGTase I inhibitor. Parallel cultures were transfected with each of the indicated Myc-Rab1B constructs. The Myc-tagged proteins were immunoprecipitated from cell lysates after an 18-h incubation with [3H]Mev in the presence or absence of 10 µM GGTI298, a specific inhibitor of GGTase I. In the upper panel one-tenth of each immunoprecipitate was subjected to immunoblot analysis. In the middle panel the remainder of the immunoprecipitate was analyzed by fluorography to visualize the prenylated proteins. The ratios of 3H (relative units) per unit of immunodetectable protein (ECL signal) are indicated in the bottom panel and are expressed as percent of the ratio determined for MycRab1B(wt) in the absence of inhibitor. Delta cys represents a MycRab1B construct that had both of the terminal cysteine residues removed.

The addition of a CLLL Motif to the C terminus of Rab1B Does Not Affect the Interaction with REP-- The absence of prenylation of Rab1B(Y78D)CLLL by REP/GGTase II in the cell-free assay (Fig. 1) suggested that the addition of CLLL motif to the Y78D mutant allowed prenylation by GGTase I but did not restore REP binding. This was confirmed in intact cells, where the CLLL versions of Myc-tagged Rab1B(wt) or Y78D were co-expressed with T7-tagged REP1. As shown in Fig. 3, size-exclusion chromatography of cytosol obtained from cells expressing MycRab1B-CLLL revealed a typical high molecular weight Rab·REP complex, similar to that previously reported for MycRab1B(wt) (23). In contrast, MycRab1B(Y78D)CLLL was unable to form a stable complex with T7-REP1, consistent with our earlier observations with MycRab1B(Y78D) containing the normal double-cysteine motif (23).


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Fig. 3.   Gel filtration analysis of MycRab1B constructs coexpressed with T7-REP in 293 cells. 293 cells were co-transfected with plasmids encoding the indicated Myc-Rab1B construct and T7-REP. The cells were maintained in medium containing 10 µM lovastatin for 48 h after transfection, then cell lysates were analyzed by gel filtration fast protein liquid chromatography, as described under "Materials and Methods." The individual fractions were subjected to SDS-PAGE and immunoblotted using monoclonal antibodies against the Myc and T7 epitopes of the expressed proteins. The graphs represent quantification of the 125I-labeled IgG from PhosphorImager analysis.

The Y78D Mutation Prevents Interaction of Rab1B with GDI-- Sequence alignments of REP and GDI reveal four major conserved regions between these two proteins (36), consistent with some functional similarities. Both proteins associate with prenylated Rab proteins in the GDP-bound conformation and can deliver them to membranes (10, 23, 26, 37, 38). However, GDI cannot replace REP in the prenylation reaction (38). For the current studies, the possibility must be considered that GDI could associate with the nascent Rab1B(Y78D)CLLL after prenylation by GGTase I and serve as an escort protein in place of REP. To address this issue, we used an established co-immune precipitation assay (26, 39) to compare the abilities of the Myc-tagged Rab constructs to interact with FLAG-GDIalpha in transfected 293 cells. Fig. 4 shows that MycRab1B and MycRab1B-CLLL were effectively co-immunoprecipitated with FLAG-GDI, whereas the Y78D mutants, regardless of the C-terminal cysteine motif, were not associated with FLAG-GDI. These observations provide direct evidence that the Switch-2 region of Rab1B is important for interaction of the GTPase with GDI as well as with REP, implying that conserved regions in GDI and REP are involved in binding the alpha 2 helix of the Rab protein. These findings are also important for the localization studies described in the following sections, because they indicate that any membrane targeting of Rab1B(Y78D)CLLL must occur by a mechanism that does not depend on REP or GDI serving as a carrier.


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Fig. 4.   Co-precipitation of Rab1B proteins with FLAG-GDI. Parallel cultures were co-transfected with plasmids encoding FLAG-GDI and one of the indicated MycRab1B constructs. Anti-FLAG affinity resin was mixed with the cell lysates to isolate proteins associated with the expressed GDI. Proteins in one-tenth of the cell lysate, before addition of the affinity resin (panel A) and the all of the eluate from the anti-FLAG resin (panel B), were subjected to SDS-PAGE and immunoblot analysis using the antibodies indicated at the left of each panel.

The Prenylated Form of Rab1B(Y78D)CLLL Can Associate with Cell Membranes-- In mammalian cells, prenylated Rab proteins are found in specific membrane compartments and in the cytosol, where they exist in a complex with GDI (10). Immunoblots from cells expressing MycRab1B and MycRab1B-CLLL showed a typical distribution of the expressed proteins between cytosol and membrane compartments (Fig. 5), consistent with their ability to undergo prenylation by one or both geranylgeranyltransferases (Figs. 1 and 2). In contrast, MycRab1B(Y78D) was localized entirely in the cytosolic fraction, as expected in light of its inability to undergo REP-dependent prenylation. Most notably, the addition of the CLLL motif to the C terminus of Y78D allowed a significant portion of the expressed protein to localize to the membrane fraction (Fig. 5).


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Fig. 5.   Subcellular fractionation of 293 cells expressing MycRab1B proteins. Parallel cultures were transfected with each of the indicated Myc-Rab1B constructs. Cell lysates were fractionated by centrifugation at 100,000 × g. The cytosolic (C) and membrane (M) fractions were immunoblotted for Myc-tagged proteins. Chemiluminescent signals, quantified by scanning with a LumiImager, are expressed as a percent of the total MycRab1B detected in both fractions.

Because earlier studies have indicated that under conditions of transient Rab overexpression a significant pool of cytosolic Rab protein remains unmodified (26), we repeated the subcellular partitioning studies, tracing the fate of [3H]Mev-labeled MycRab1B instead of total MycRab1B (Fig. 6). As expected, no [3H]Mev-labeled protein was detected when MycRab1B(Y78D) was immunoprecipitated from the soluble or membrane fractions of transfected 293 cells. However, when the same protein was converted to a substrate for GGTase I by the addition of the CLLL motif, radiolabeled protein was clearly detected. The partitioning of the prenylated Rab1B(Y78D)CLLL resembled that of the wild-type Rab1B, with the majority of the [3H]Mev-labeled protein present in the membrane fraction. Interestingly, these studies also revealed that changing the C terminus of wild-type Rab1B from CC to CLLL caused a significant shift in its subcellular distribution. Specifically, the percent of total [3H]Mev-labeled protein in the cytosolic fraction increased from less than 10% in the case of Rab1B to more than 65% in the case of Rab1B-CLLL (Fig. 6). A possible explanation for the increased proportion of prenylated Rab1B-CLLL in the cytosol is provided by the studies of Shen and Seabra (40), who reported that mono-geranylgeranylated Rab1A forms a more stable complex with REP than the di-geranylgeranylated protein. For Rabs with a double-cysteine motif, this presumably allows the mono-prenylated intermediate to remain tightly associated with REP in the cytosol until geranylgeranylation of the second cysteine is complete. The lower affinity of the doubly-prenylated form of the Rab for REP would then allow the GTPase to dissociate from the escort protein when an acceptor membrane is encountered. In the context of this model, we would expect to see an increased cytosolic pool of mono-prenylated Rab1B-CLLL but not Rab1B(Y78D)CLLL, since the latter cannot interact with REP and is prenylated exclusively by GGTase I. 


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Fig. 6.   Subcellular distribution of prenylated Myc-Rab1B proteins. Parallel cultures were transfected with each of the indicated Myc-Rab1B constructs. The cells were incubated with [3H]Mev for 18 h, then the Myc-tagged proteins were immunoprecipitated from membrane and cytosolic fractions. The upper panel shows the fluorograph of the immunoprecipitated proteins to visualize the prenylation. The bottom panel is a graph of the data quantified by densitometer analysis. C, cytosolic fractions; M, membrane fractions.

Immunofluorescent Localization of Rab1B in Intracellular Membranes-- From the preceding observations we conclude that MycRab1B(Y78D)CLLL can associate with membranes after prenylation by GGTase I in intact cells. This apparently occurs through a mechanism that does not require the formation of a REP or GDI carrier complex. We next carried out a study using immunofluorescence microscopy to determine whether or not the CLLL-modified versions of Rab1B were delivered to the same subcellular compartments as the wild-type Rab1B. Cells expressing MycRab1B(wt) exhibited a juxta-nuclear staining pattern that showed significant overlap with proteins known to function in the Golgi compartment; i.e. GM130 (41) and Rab6 (42) (Fig. 7). MycRab1B(wt) also showed partial co-localization with a resident ER protein, calreticulin (43) (Fig. 7). This is typical of the localization reported for Rab1 in previous studies (5, 6, 26).


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Fig. 7.   Immunofluorescent localization of MycRab1B. 293 cells were transfected with the expression vector encoding MycRab1B(wt). The next day the cells were fixed and co-stained with a Myc antibody and the indicated Golgi (Rab6 or GM130) or ER marker (calreticulin) antibody, followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG as described under "Materials and Methods." Gray scale images were pseudo-colored to correspond to the red (rhodamine) and green (fluorescein isothiocyanate) fluorescence, with yellow indicating the overlapping regions.

To directly compare the localization of the MycRab1B-CLLL constructs with the localization of the wild-type protein in the same cells, we carried out a co-transfection study in which Rab1B(wt) was tagged with the HA epitope and the CLLL constructs were tagged with the Myc epitope. Preliminary studies were first performed in which cells were co-transfected with Rab1B(wt) constructs bearing the different epitope tags. These studies established that the localization pattern of Rab1B was not changed by the HA epitope (not shown). The images in Fig. 8 demonstrate that MycRab1B-CLLL (mono-prenylated but still competent to bind REP) had an immunofluorescent localization pattern nearly identical to that of HA-Rab1B(wt). The localization pattern of MycRab1B(Y78D)CLLL (mono-prenylated, but incompetent to bind REP) was similar to that of HA-Rab1B(wt) but also contained a peripheral component that extended beyond the perinuclear region in a pattern reminiscent of calreticulin. From these observations we can conclude that MycRab1B(wt), MycRab1B-CLLL, and MycRab1B(Y78D)CLLL were all targeted to ER/Golgi membranes, although the latter construct must arrive in this compartment by a mechanism that does not depend on REP or GDI escort functions.


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Fig. 8.   Co-localization of CLLL mutants with HA-Rab1B(wt). 293 cells were co-transfected with expression vectors encoding HA-Rab1B(wt) and either MycRab1B-CLLL or MycRab1B(Y78D)CLLL. The next day the cells were fixed and co-stained with Myc polyclonal and HA monoclonal antibodies followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG as described under "Materials and Methods." Gray scale images were pseudo-colored to correspond to the red (rhodamine) and green (fluorescein isothiocyanate) fluorescence, with yellow indicating the overlapping regions.

MycRab1B-CLLL and MycRab1B(Y78D)CLLL Undergo C-terminal Proteolytic Processing and Carboxymethylation in HEK293 Cells-- Proteins modified by GGTase I typically undergo additional modifications consisting of the removal of the three amino acids distal to the prenylated cysteine followed by C-terminal carboxymethylation of the exposed prenyl cysteine residue (44). The results shown in Fig. 9 demonstrate that volatile [3H]methyl groups were incorporated into the immunoprecipitated MycRab1B-CLLL and MycRab1B(Y78D)CLLL when these proteins were expressed in 293 cells incubated with [methyl-3H]methionine. In contrast, no specific incorporation was detected in MycRab1B or MycRab1B(Y78D), which terminate with a typical Rab double-cysteine motif (CC) that does not undergo carboxymethylation (45, 46). Accumulating evidence indicates that the prenyl-cysteine carboxymethyltransferase resides in the ER (47, 48). Therefore, these findings provide additional evidence that Rab1B(Y78D)CLLL was delivered to membranes of the ER, independent of interaction with REP or GDI.


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Fig. 9.   Carboxymethylation of MycRab1B proteins expressed in 293 cells. Parallel cultures were transfected with each of the indicated MycRab1B constructs. The cells were incubated with [methyl-3H]methionine for 19 h, then the Myc-tagged proteins were immunoprecipitated (IP) and separated by SDS-PAGE. The dried gel lanes were cut into 0.5-cm sections, and a vapor-phase equilibration assay was used to measure the tritium incorporation into base-hydrolyzable methyl esters. The graphs show the amount of volatile tritium released from the gel slices. The upper left panel is an immunoblot showing the MycRab1B collected in one-tenth of each immunoprecipitate. The arrow at the top of each panel marks the gel slice containing the immunodetectable MycRab1B.

The Y78D Mutation Abrogates the Activity of a Dominant-negative Rab1B Mutant-- To determine whether the mono-prenylated Rab1B constructs bearing the CLLL motif and/or the Y78D substitution might interfere with ER right-arrow Golgi transport, we co-expressed these proteins together with the human low density lipoprotein receptor (LDLR) in 293 cells. The LDLR undergoes O-glycosylation in the medial Golgi compartment, resulting in a shift in its electrophoretic mobility on SDS gels from a sharp band at ~120 kDa (the immature, ER form) to a poorly resolved band between 160 and 170 kDa (32, 49, 50). As shown in Fig. 10, when either Rab1B(wt), Rab1B-CLLL, Rab1B(Y78D), or Rab1B(Y78D)CLLL was co-expressed with the LDLR, processing of the radiolabeled receptor to the mature O-glycosylated form was readily detected by pulse-chase analysis of the immunoprecipitated protein. Thus, the Y78D constructs do not impair the function of endogenous Rab1-dependent ER right-arrow Golgi transport pathways. Previous studies have shown that Rab proteins bearing amino acid substitutions at the position equivalent to Ser-17 in Ha-Ras are locked in the inactive GDP state because they have a greatly reduced affinity for GTP but not GDP (6, 7, 51). Introduction of such mutations into Rab1A (S25N) or Rab1B(S22N) causes these proteins to act as dominant suppressors of Rab1 function in cultured cells, so that ER right-arrow Golgi protein trafficking is arrested (6). To determine whether the mono-prenylated form of Rab1B (S22N) would still be able to suppress ER right-arrow Golgi trafficking, we converted its C terminus to the CLLL motif. As shown in Fig. 10, the mono-prenylated Rab1B(S22N)CLLL suppressed LDLR processing just as well as the Rab1B(S22N) with the normal CC motif (Fig. 10). However, when the Y78D mutation was inserted into the Rab1B(S22N)CLLL construct, the dominant-negative effect of the S22N mutation on ER right-arrow Golgi trafficking of the LDLR was lost. Thus, even though MycRab1B-CLLL and MycRab1B(Y78D)CLLL show similar localization (Fig. 8), only the former construct suppresses ER right-arrow Golgi trafficking when the S22N substitution is introduced.


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Fig. 10.   Effect of expression of various MycRab1B constructs on LDL receptor processing. The indicated MycRab1B constructs were co-expressed with the LDLR in 293 cells. 24 h after transfection, the cells were pulse-labeled for 30 min with [35S]Met, then chased with excess cold methionine and cysteine for 2 h. Immunoprecipitation of the LDLR was analyzed by SDS-PAGE and fluorography. m indicates the migration position of the mature O-glycosylated LDLR, whereas i indicates the position of the immature form of the receptor found in the ER.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we have examined the requirement for REP in the membrane delivery of Rab1B in intact cells by using four different Myc-tagged Rab1B constructs as follows. (i) Rab1B(wt) binds to REP and is di-geranylgeranylated exclusively by GGTase II. (ii) Rab1B(Y78D) cannot bind REP and therefore fails to undergo prenylation. (iii) Rab1B-CLLL binds to REP and is geranylgeranylated on the single cysteine by GGTase II, but it can also be modified in a REP-independent manner by GGTase I. (iv) Rab1B(Y78D)CLLL cannot bind REP but can be geranylgeranylated by GGTase I. Our results show that whereas Rab1B(Y78D) partitioned exclusively in the cytosol, both Rab1B-CLLL and Rab1B(Y78D)CLLL accumulated in membrane fractions of transfected cells (Figs. 5 and 6). The subcellular distribution patterns of these proteins resembled that of the wild-type Rab1B (Figs. 7 and 8). Moreover, they underwent additional modifications typical of CAAX-motif proteins (proteolytic removal of the terminal LLL tripeptide and carboxymethylation, Fig. 9) that require enzymes localized in the ER (48, 52). It is well established that under normal circumstances both REP and GDI function in the delivery of prenylated Rab proteins to intracellular membranes (38, 40, 53, 54). However, since the Y78D mutation prevents Rab1B from binding to REP or GDI (Figs. 3 and 4) (23), we conclude that protein interactions mediated by these carrier proteins are not absolutely required for targeting of Rab1B to intracellular membranes, provided that the C terminus can be geranylgeranylated by an alternative mechanism (GGTase I).

It remains to be determined precisely how the mono-geranylgeranylated form of Rab1B(Y78D)CLLL is delivered to membranes in the absence of REP or GDI. However, it seems likely that the initial targeting of this protein to the ER may follow the same path as newly prenylated Ras and Rho GTPases. In this regard, the recent studies of Dai and co-workers (48) suggest a model wherein the prenylated cysteine, in the context of the CAAX motif, may serve as a signal structure that targets proteins to a receptor on the ER membrane, so that AAX-trimming and carboxymethylation of different GTPases can be completed by a common set of enzymes. Proteins destined for peripheral membranes (e.g. Ras, RhoA, Rac1) are then sorted by mechanisms yet to be defined (55), whereas proteins that normally function in the endomembrane compartment (in this case, Rab1B) remain there.

The recent studies of Allan et al. (56) provide insights into the role of Rab1 in the vesicle budding and fusion machinery of ER right-arrow Golgi transport. Specifically, it appears that Rab1 in its active GTP-bound state may recruit a tethering protein, p115, to the coat protein complex II (COPII) on budding ER vesicles. The GTPase then promotes the assembly of a functional SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) complex required for vesicle fusion with the Golgi acceptor compartment (56). An important question raised by the present studies is whether the functional interaction of Rab1B with the COPII complex is affected by the specific nature of the post-translational modifications (di-geranylgeranylation versus mono-geranylgeranylation and carboxymethylation) or the mode of delivery of Rab1B to the ER/Golgi membranes (REP-mediated versus prenyl CAAX-mediated translocation from GGTase I). We approached this question by examining the effects of the C-terminal CLLL modification and Y78D substitution on the activity of the dominant-negative mutant, Rab1B(S22N), in intact cells. The biological activity of this mutant depends on prenylation (7) and presumably involves competition with endogenous Rab1 for binding to nucleotide exchange factors or other docking proteins in the COPII complex on the budding transport vesicle (7, 57, 58). Our results show that Rab1B(S22N)CLLL is fully capable of suppressing ER right-arrow Golgi transport of the LDL receptor (Fig. 10), implying that the relevant Rab1B(S22N) protein interactions can be supported by mono-geranylgeranylation and carboxymethylation instead of di-geranylgeranylation. However, when the Y78D mutation was introduced into Rab1B(S22N)CLLL, its inhibitory effect on LDL receptor trafficking was lost.

There are two obvious possibilities that could account for the ability of the Y78D mutation to eliminate the inhibitory activity of Rab1B(S22N). The first is that REP (or GDI) plays a cooperative role with the Rab1 GTPase to promote its interaction with the COPII docking complex or nucleotide exchange factors at the vesicle membrane. According to this model, the Y78D mutation, by preventing Rab1B from associating with REP (23) or GDI (Fig. 4), indirectly prevents the dominant-negative mutant from binding to its protein targets even though it can reach the ER membrane by virtue of the prenylated CAAX motif. Although studies of the yeast REP, Mrs6p, suggest that REP may indeed bind to proteins in ER/Golgi membranes (59), there is presently no direct evidence that cooperative interactions mediated by REP are absolutely required for functional Rab1B association with the COPII complex. On the contrary, in vitro experiments have shown that recombinant Rab1B can support ER right-arrow Golgi transport when added to perforated cells in monomeric form rather than as a REP or GDI complex (4). The alternative explanation for the loss of inhibitory activity that occurs when the Y78D mutation is combined with the S22N mutation would postulate that the alpha 2 helix in the predicted Switch-2 region of Rab1B is not only critical for interaction with REP and GDI but is also involved in the association of Rab1B with docking proteins or exchange factors on the budding transport vesicle. Support for this view comes from earlier studies of chimeric Rab proteins showing that the alpha 2 helix of Rab5 is one of the domains that affects its localization and functional specificity (60). Thus, it will be interesting to determine how amino acid substitutions in the alpha 2 helix of Rab1B may affects its ability to interact with components of the COPII complex. The functional evaluation of such Rab mutants has been hindered by the fact that they cannot be prenylated and delivered to membranes by the REP-dependent GGTase II pathway. However, the present study demonstrates that this problem can by circumvented by changing the C-terminal motif to one that can be recognized by GGTase I.

    ACKNOWLEDGEMENT

We are grateful to Miguel Seabra for providing purified REP and GGTase II, Patrick Casey for providing purified GGTase I, and Said Sebti for providing GGTI-298. We also thank Deborah Heitzman for technical assistance with preparation of the HA-tagged Rab constructs.

    FOOTNOTES

To whom correspondence should be addressed: Dept of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-4100; Fax: 419-383-6228; E-mail: wmaltese@mco.edu

Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M101511200

This work was supported by National Institutes of Health Grant CA34569 (to W. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2 CAAL represents a cysteine residue followed by two aliphatic amino acids and a terminal leucine residue.

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; GDI, guanine nucleotide dissociation inhibitor; GGTase, geranylgeranyltransferase; REP, Rab escort protein; HEK, human embryonal kidney; Mev, mevalonate; LDL, low density lipoprotein; LDLR, LDL receptor; COPII, coat protein complex II; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Chavrier, P., Simons, K., and Zerial, M. (1992) Gene (Amst.) 112, 261-264[Medline] [Order article via Infotrieve]
2. Novick, P., and Zerial, M. (1997) Curr. Opin. Cell Biol. 9, 496-504[CrossRef][Medline] [Order article via Infotrieve]
3. Pfeffer, S. R. (1994) Curr. Opin. Cell Biol. 6, 522-526[Medline] [Order article via Infotrieve]
4. Plutner, H., Cox, A. D., Pind, S., Khosravi-Far, R., Bourne, J., Schwaninger, R., Der, C. J., and Balch, W. E. (1991) J. Cell Biol. 115, 31-43[Abstract]
5. Saraste, J., Lahtinen, U., and Goud, B. (1995) J. Cell Sci. 108, 1541-1552[Abstract/Free Full Text]
6. Tisdale, E. J., Bourne, J. R., Khosravi-Far, R., Der, C. J., and Balch, W. E. (1992) J. Cell Biol. 119, 749-761[Abstract]
7. Nuoffer, C., Davidson, H. W., Matteson, J., Meinkoth, J., and Balch, W. E. (1994) J. Cell Biol. 125, 225-237[Abstract]
8. Nuoffer, C., and Balch, W. E. (1994) Annu. Rev. Biochem. 63, 949-990[CrossRef][Medline] [Order article via Infotrieve]
9. Martinez, O., and Goud, B. (1998) Biochim. Biophys. Acta 1404, 101-112[Medline] [Order article via Infotrieve]
10. Pfeffer, S. R., Dirac-Svejstrup, A. B., and Soldati, T. (1995) J. Biol. Chem. 270, 17057-17059[Free Full Text]
11. Lupashin, V. V., and Waters, M. G. (1997) Science 276, 1255-1258[Abstract/Free Full Text]
12. Rothman, J. E., and Sollner, T. H. (1997) Science 276, 1212-1213[Free Full Text]
13. Khosravi-Far, R., Lutz, R. J., Cox, A. D., Conroy, L., Bourne, J. R., Sinensky, M., Balch, W. E., Buss, J. E., and Der, C. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6264-6268[Abstract]
14. Overmeyer, J. H., and Maltese, W. A. (1992) J. Biol. Chem. 267, 22686-22692[Abstract/Free Full Text]
15. Musha, T., Kawata, M., and Takai, Y. (1992) J. Biol. Chem. 267, 9821-9825[Abstract/Free Full Text]
16. Soldati, T., Riederer, M. A., and Pfeffer, S. R. (1993) Mol. Biol. Cell 4, 425-434[Abstract]
17. Seabra, M. C., Goldstein, J. L., Sudhof, T. C., and Brown, M. S. (1992) J. Biol. Chem. 267, 14497-14503[Abstract/Free Full Text]
18. Casey, P. J., and Seabra, M. C. (1996) J. Biol. Chem. 271, 5289-5292[Free Full Text]
19. Andres, D. A., Seabra, M. C., Brown, M. S., Armstrong, S. A., Smeland, T. E., Cremers, F. P. M., and Goldstein, J. L. (1993) Cell 73, 1091-1099[Medline] [Order article via Infotrieve]
20. Alexandrov, K., Simon, I., Yurchenko, V., Iakovenko, A., Rostkova, E., Scheidig, A. J., and Goody, R. S. (1999) Eur. J. Biochem. 265, 160-170[Abstract/Free Full Text]
21. Seabra, M. C. (1996) J. Biol. Chem. 271, 14398-14404[Abstract/Free Full Text]
22. Chavrier, P., Gorvel, J.-P., Stelzer, E., Simons, K., Gruenberg, J., and Zerial, M. (1991) Nature 353, 769-772[CrossRef][Medline] [Order article via Infotrieve]
23. Overmeyer, J. H., Wilson, A. L., Erdman, R. A., and Maltese, W. A. (1998) Mol. Biol. Cell 9, 223-235[Abstract/Free Full Text]
24. Wilson, A. L., Sheridan, K. M., Erdman, R. A., and Maltese, W. A. (1996) Biochem. J. 318, 1007-1014[Medline] [Order article via Infotrieve]
25. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H., and Russell, D. W. (1989) J. Biol. Chem. 264, 8222-8229[Abstract/Free Full Text]
26. Wilson, A. L., Erdman, R. A., and Maltese, W. A. (1996) J. Biol. Chem. 271, 10932-10940[Abstract/Free Full Text]
27. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
28. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
29. Vogt, A., Qian, Y., McGuire, T., Hamilton, A. D., and Sebti, S. M. (1996) Oncogene 13, 1991-1999[Medline] [Order article via Infotrieve]
30. Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647[Abstract]
31. Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688[Abstract/Free Full Text]
32. Castellano, F. C., Wilson, A. L., and Maltese, W. A. (1995) J. Recept. Signal Transduct. Res. 15, 847-862[Medline] [Order article via Infotrieve]
33. Wilson, A. L., Erdman, R. A., Castellano, F., and Maltese, W. A. (1998) Biochem. J. 333, 497-504[Medline] [Order article via Infotrieve]
34. Kinsella, B. T., Erdman, R. A., and Maltese, W. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8934-8938[Abstract]
35. Casey, P. J., Thissen, J. A., and Moomaw, J. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8631-8635[Abstract]
36. Schalk, I., Zeng, K., Wu, S.-K., Stura, E., Matteson, J., Huang, M., Tandon, A., Wilson, I. A., and Balch, W. E. (1996) Nature 381, 42-48[CrossRef][Medline] [Order article via Infotrieve]
37. Ullrich, O., Stenmark, H., Alexandrov, K., Hubar, L. A., Kaibuchi, K., Sasaki, T., Takai, Y., and Zerial, M. (1993) J. Biol. Chem. 268, 18143-18150[Abstract/Free Full Text]
38. Alexandrov, K., Horiuchi, H., Steele-Mortimer, O., Seabra, M. C., and Zerial, M. (1994) EMBO J. 13, 5262-5273[Abstract]
39. Erdman, R. A., Shellenberger, K. E., Overmeyer, J. H., and Maltese, W. A. (2000) J. Biol. Chem. 275, 3848-3856[Abstract/Free Full Text]
40. Shen, F., and Seabra, M. C. (1996) J. Biol. Chem. 271, 3692-3698[Abstract/Free Full Text]
41. Nakamura, N., Rabouille, C., Watson, R., Nilsson, T., Hui, N., Slusarewicz, P., Kreis, T. E., and Warren, G. (1995) J. Cell Biol. 131, 1715-1726[Abstract]
42. Antony, C., Cibert, C., Geraud, G., Santa Maria, A., Maro, B., Mayau, V., and Goud, B. (1992) J. Cell Sci. 103, 785-796[Abstract/Free Full Text]
43. Danilczyk, U. G., Cohen-Doyle, M. F., and Williams, D. B. (2000) J. Biol. Chem. 275, 13089-13097[Abstract/Free Full Text]
44. Clarke, S. (1992) Annu. Rev. Biochem. 61, 355-386[CrossRef][Medline] [Order article via Infotrieve]
45. Wei, C., Lutz, R., Sinensky, M., and Macara, I. G. (1992) Oncogene 7, 467-473[Medline] [Order article via Infotrieve]
46. Newman, C. M. H., Giannakouros, T., Hancock, J. F., Fawell, E. H., Armstrong, J., and Magee, A. I. (1992) J. Biol. Chem. 267, 11329-11336[Abstract/Free Full Text]
47. Jang, G. F., Yokoyama, K., and Gelb, M. H. (1993) Biochemistry 32, 9500-9507[Medline] [Order article via Infotrieve]
48. Dai, Q., Choy, E., Chiu, V., Romano, J., Slivka, S. R., Steitz, S. A., Michaelis, S., and Philips, M. R. (1998) J. Biol. Chem. 273, 15030-15034[Abstract/Free Full Text]
49. Tolleshaug, H., Goldstein, J. L., Schneider, W., and Brown, M. S. (1982) Cell 30, 715-724[Medline] [Order article via Infotrieve]
50. Cummings, R. D., Kornfeld, S., Schneider, W. J., Hobgood, K. K., Tolleshaug, H., Brown, M. S., and Goldstein, J. L. (1983) J. Biol. Chem. 258, 15261-15273[Abstract/Free Full Text]
51. Barbieri, M. A., Li, G., Columbo, M. I., and Stahl, P. D. (1994) J. Biol. Chem. 269, 18720-18722[Abstract/Free Full Text]
52. Schmidt, W. K., Tam, A., Fujimura-Kamada, K., and Michaelis, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11175-11180[Abstract/Free Full Text]
53. Wu, S.-K., Luan, P., Matteson, J., Zeng, K., Nishimura, N., and Balch, W. E. (1998) J. Biol. Chem. 273, 26931-26938[Abstract/Free Full Text]
54. Ullrich, O., Horiuchi, H., Bucci, C., and Zerial, M. (1994) Nature 368, 157-160[CrossRef][Medline] [Order article via Infotrieve]
55. Michaelson, D., Silletti, J., Murphy, G., D'Eustachio, P., Rush, M., and Philips, M. R. (2001) J. Cell Biol. 152, 111-126[Abstract/Free Full Text]
56. Allan, B. B., Moyer, B. D., and Balch, W. E. (2000) Science 289, 444-448[Abstract/Free Full Text]
57. Pind, S. N., Nuoffer, C., McCaffery, J. M., Plutner, H., Davidson, H. W., Farquhar, M. G., and Balch, W. E. (1994) J. Cell Biol. 125, 239-252[Abstract]
58. Dirac-Svejstrup, A. B., Sumizawa, T., and Pfeffer, S. R. (1997) EMBO J. 16, 465-472[Abstract/Free Full Text]
59. Miaczynska, M., Lorenzetti, S., Bialek, U., Benito-Moreno, R. M., Schweyen, R. J., and Ragnini, A. (1997) J. Biol. Chem. 272, 16972-16977[Abstract/Free Full Text]
60. Stenmark, H., Valencia, A., Martinez, O., Ullrich, O., Goud, B., and Zerial, M. (1994) EMBO J. 13, 575-583[Abstract]


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