Nucleotide Dependence of Rab Geranylgeranylation

Geranylgeranylation of Rab GTPases is an essential post-translational modification that enables Rabs to associate with intracellular membranes where they regulate exocytic and endocytic pathways. Geranylgeranylation is initiated by formation of a stable complex between newly synthesized Rab proteins and Rab escort protein (REP). The complex is recognized by Rab geranylgeranyl (GG) transferase, which transfers two GG groups to Rabs. The geranylgeranylated Rabs regulate vesicular movement by oscillating between an inactive GDP-bound form and an active GTP-bound form. In this study, I show that the kinetics of geranylgeranylation is influenced by the nucleotide status of nascent Rab. GDP-bound Rab is geranylgeranylated with 10–50-fold higher affinity than GTP-bound Rab (or GTP analog-bound Rab), as indicated by the apparent Km of the reaction. In vitro REP·Rab binding assays demonstrate that REP forms a stable complex only with the GDP-bound form of Rab but not the GTP-bound form, suggesting that the apparent Km effect in the prenylation reaction is due to a discrimination between the two different nucleotide-bound forms of Rab by REP. Inasmuch as Rabs are likely GTP-bound after synthesis and REP does not possess GTPase-activating protein activity, these results raise the possibility that a Rab GTPase-activating protein enhances the REP·Rab interaction prior to prenylation. Geranylgeranylation of Rab GTPases is an essential post-translational modification that enables Rabs to associate with intracellular membranes where they regulate exocytic and endocytic pathways. Geranylgeranylation is initiated by formation of a stable complex between newly synthesized Rab proteins and Rab escort protein (REP). The complex is recognized by Rab geranylgeranyl (GG) transferase, which transfers two GG groups to Rabs. The geranylgeranylated Rabs regulate vesicular movement by oscillating between an inactive GDP-bound form and an active GTP-bound form. In this study, I show that the kinetics of geranylgeranylation is influenced by the nucleotide status of nascent Rab. GDP-bound Rab is geranylgeranylated with 10–50-fold higher affinity than GTP-bound Rab (or GTP analog-bound Rab), as indicated by the apparent Km of the reaction. In vitro REP·Rab binding assays demonstrate that REP forms a stable complex only with the GDP-bound form of Rab but not the GTP-bound form, suggesting that the apparent Km effect in the prenylation reaction is due to a discrimination between the two different nucleotide-bound forms of Rab by REP. Inasmuch as Rabs are likely GTP-bound after synthesis and REP does not possess GTPase-activating protein activity, these results raise the possibility that a Rab GTPase-activating protein enhances the REP·Rab interaction prior to prenylation. INTRODUCTIONPost-translational modification of proteins with lipids is a frequent and important cellular regulatory mechanism that has only recently been appreciated (reviewed in 1Resh M.D. Cell. 1994; 76: 411-413Google Scholar, 2Milligan G. Parenti M. Magee A.I. Trends Biochem. Sci. 1995; 20: 181-187Google Scholar, 3Casey P.J. Science. 1995; 268: 221-225Google Scholar, 4Glomset J.A. Farnsworth C.C. Annu. Rev. Cell Biol. 1994; 10: 181-205Google Scholar). The lipid moieties, including acyl groups, prenyl groups, or both, are essential for the function of the modified protein, because they serve as determinants for protein-membrane or protein-protein interactions. Two types of prenyl groups have been identified to date: farnesyl or geranylgeranyl (GG). 1The abbreviations used are: GGgeranylgeranylGGPPgeranylgeranyl pyrophosphateREPRab escort proteinGGTasegeranylgeranyl transferaseGMPPNPguanosine 5′-(β,γ-imido)triphosphateGDIGDP dissociation inhibitorGAPGTPase-activating protein. Both are attached to carboxyl-terminal cysteine residues via thioether bonds.Protein prenylation is catalyzed by three cytosolic enzymes that can be classified in two groups: the “CAAX” prenyl transferases, which include farnesyl transferase and GG transferase-I, and the Rab GG transferase or GG transferase-II (5Casey P.J. Seabra M.C. J. Biol. Chem. 1996; 271: 5289-5292Google Scholar). CAAX prenyl transferases recognize a distinct motif at the carboxyl termini of their intracellular substrates. The motif is designated CAAX, where C is cysteine, A is aliphatic, and X is any amino acid. The CAAX sequence is the primary determinant for binding to the protein substrate. If the last amino acid in the CAAX sequence is a methionine or serine, the protein is a substrate of farnesyl transferase, whereas if it is a leucine it becomes a substrate for GGTase-I (6Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Google Scholar, 7Seabra M.C. Reiss Y. Casey P.J. Brown M.S. Goldstein J.L. Cell. 1991; 65: 429-434Google Scholar, 8Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'Hara M.B. Garsky V.M. Marshall M.S. Pompliano D.L. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Google Scholar, 9Moomaw J.F. Casey P.J. J. Biol. Chem. 1992; 267: 17438-17443Google Scholar, 10Yokoyama K. Gelb M.H. J. Biol. Chem. 1993; 268: 4055-4060Google Scholar). CAAX-containing proteins include members of the Ras and Rho/Rac family of low molecular weight GTPases, γ-subunits of G-proteins, nuclear lamins, G-protein-coupled receptor kinases, and retinal cyclic GMP phosphodiesterase.All proteins belonging to a large subfamily of low molecular weight GTPases, termed Rab in mammals and YPT/SEC4 in yeast, undergo geranylgeranylation even though they lack a classical CAAX motif at the carboxyl terminus. Instead, these proteins typically possess a double-cysteine motif, such as CC or CXC, of which both cysteines are modified by GG groups (11Farnsworth C.C. Kawata M. Yoshida Y. Takai Y. Gelb M.H. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6196-6200Google Scholar, 12Farnsworth C.C. Seabra M.C. Ericsson L.H. Gelb M.H. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11963-11967Google Scholar). An enzyme that digeranylgeranylates Rab proteins has been purified and molecularly cloned from rat brain and characterized in in vitro prenylation assays (12Farnsworth C.C. Seabra M.C. Ericsson L.H. Gelb M.H. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11963-11967Google Scholar, 13Seabra M.C. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1992; 267: 14497-14503Google Scholar, 14Seabra M.C. Brown M.S. Slaughter C.A. Sudhof T.C. Goldstein J.L. Cell. 1992; 70: 1049-1057Google Scholar, 15Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P.M. Goldstein J.L. Cell. 1993; 73: 1091-1099Google Scholar, 16Armstrong S.A. Seabra M.C. Sudhof T.C. Goldstein J.L. Brown M.S. J. Biol. Chem. 1993; 268: 12221-12229Google Scholar, 17Cremers F.P.M. Armstrong S.A. Seabra M.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1994; 269: 2111-2117Google Scholar). This enzyme, called Rab GGTase or GGTase-II (previously designated Component B of Rab GGTase), is a heterodimer composed of an α-subunit and a β-subunit, both homologous to the corresponding α- and β-subunits of the CAAX prenyl transferases (13Seabra M.C. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1992; 267: 14497-14503Google Scholar, 16Armstrong S.A. Seabra M.C. Sudhof T.C. Goldstein J.L. Brown M.S. J. Biol. Chem. 1993; 268: 12221-12229Google Scholar). However, in contrast with the CAAX prenyl transferases, Rab GGTase does not recognize Rab substrates unless they are complexed with another cytosolic protein, designated Rab Escort Protein (REP), previously designated Component A of Rab GGTase. REP is also known as the choroideremia gene product (see below) (14Seabra M.C. Brown M.S. Slaughter C.A. Sudhof T.C. Goldstein J.L. Cell. 1992; 70: 1049-1057Google Scholar, 15Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P.M. Goldstein J.L. Cell. 1993; 73: 1091-1099Google Scholar, 17Cremers F.P.M. Armstrong S.A. Seabra M.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1994; 269: 2111-2117Google Scholar).Previous studies suggested that newly synthesized Rabs bind REP, and the complex is then recognized by Rab GGTase. Two consecutive GG additions occur, possibly starting with the cysteine furthest from the carboxyl terminus. The product of the reaction, diGG-Rab·REP complex, likely dissociates after translocation of the diGG-Rab into a specific intracellular membrane (15Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P.M. Goldstein J.L. Cell. 1993; 73: 1091-1099Google Scholar, 18Alexandrov K. Horiuchi H. Steele-Mortimer O. Seabra M.C. Zerial M. EMBO J. 1994; 13: 5262-5273Google Scholar, 19Shen F. Seabra M.C. J. Biol. Chem. 1996; 271: 3692-3699Google Scholar). Alternatively, it has been proposed that Rab GDP dissociation inhibitor (Rab GDI) serves as a cytosolic acceptor for diGG-Rabs by promoting the dissociation of the diGG-Rab·REP complex (20Sanford J.C. Yu J. Pan J.Y. Wessling-Resnick M. J. Biol. Chem. 1995; 270: 26904-26909Google Scholar). REP is absolutely required for in vitro prenylation of Rab proteins, and studies in yeast and humans suggest that it is also required in vivo. In Saccharomyces cerevisiae, the REP homolog MRS6/MSI4 is an essential gene (21Waldherr M. Ragnini A. Schweyer R.J. Boguski M.S. Nat. Genet. 1993; 3: 193-194Google Scholar, 22Fujimura K. Tanaka K. Nakano A. Toh-e A. J. Biol. Chem. 1994; 269: 9205-9212Google Scholar). In mammals, there are two known REPs, REP-1 and REP-2, and in humans, loss-of-function mutations in the REP-1 gene result in choroideremia, an X-linked form of retinal degeneration (14Seabra M.C. Brown M.S. Slaughter C.A. Sudhof T.C. Goldstein J.L. Cell. 1992; 70: 1049-1057Google Scholar, 23Cremers F.P.M. van de Pol D.J. van Kerkhoff L.P. Wieringa B. Ropers H.H. Nature. 1990; 347: 674-677Google Scholar, 24Seabra M.C. Brown M.S. Goldstein J.L. Science. 1993; 259: 377-381Google Scholar). Evidence suggests that REP-1 and REP-2 are almost entirely functionally redundant, at least at the level of substrate specificity (15Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P.M. Goldstein J.L. Cell. 1993; 73: 1091-1099Google Scholar, 17Cremers F.P.M. Armstrong S.A. Seabra M.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1994; 269: 2111-2117Google Scholar, 25Seabra M.C. Ho Y.K. Anant J.S. J. Biol. Chem. 1995; 270: 24420-24427Google Scholar, 26Cremers F.P.M. Molloy C.M. van de Pol D.J. van den Hurk J.A. Bach I. Geurts van Kessel A.H. Ropers H.H. Hum. Mol. Genet. 1992; 1: 71-75Google Scholar). In mutant choroideremia lymphoblastoid cells presumably all Rab prenylation is carried out by REP-2; only one Rab protein, Rab27, could be detected in an unprenylated form (25Seabra M.C. Ho Y.K. Anant J.S. J. Biol. Chem. 1995; 270: 24420-24427Google Scholar). Because Rab27 is expressed in the two retinal cell types that degenerate primarily in choroideremia, the retinal pigment epithelium and the choroid, we proposed that the pathogenesis of choroideremia may be related to Rab27 dysfunction (25Seabra M.C. Ho Y.K. Anant J.S. J. Biol. Chem. 1995; 270: 24420-24427Google Scholar).Once prenylated, Rabs associate with the cytoplasmic leaflet of the membrane surrounding a nascent transport vesicle. There, Rabs are proposed to regulate the targeting and fusion of transport vesicles to the appropriate acceptor compartments in exocytic and endocytic pathways (27Zerial M. Stenmark H. Curr. Opin. Cell Biol. 1993; 5: 613-620Google Scholar, 28Pfeffer S.R. Dirac-Svejstrup A.B. Soldati T. J. Biol. Chem. 1995; 270: 17057-17059Google Scholar, 29Nuoffer C. Balch W.E. Annu. Rev. Biochem. 1994; 63: 949-990Google Scholar). Like other GTP-binding proteins, Rabs cycle between inactive GDP-bound and active GTP-bound forms. Rabs associate with membranes in the GDP-bound form and switch to a GTP-bound conformation in a reaction catalyzed by a GDP/GTP exchange factor (30Soldati T. Shapiro A.D. Dirac-Svejstrup A.B. Pfeffer S.R. Nature. 1994; 369: 76-78Google Scholar, 31Ullrich O. Horiuchi H. Bucci C. Zerial M. Nature. 1994; 368: 157-160Google Scholar, 32Horiuchi H. Giner A. Hoflack B. Zerial M. J. Biol. Chem. 1995; 270: 11257-11262Google Scholar). After fusion has occurred, the GTP is hydrolyzed in a reaction that is stimulated by a Rab-specific GTPase-activating protein (Rab GAP). This reaction forces Rabs into the GDP-bound form, which leads to their removal from the membrane by Rab GDI. In response to subsequent signals, Rab GDI mediates the reassociation of GDP-Rab with the donor membrane, where the cycle resumes. Rab GDI and REP share structural and functional properties, and it has been proposed that REP acts in the delivery of newly synthesized Rabs, whereas Rab GDI acts in their recycling (18Alexandrov K. Horiuchi H. Steele-Mortimer O. Seabra M.C. Zerial M. EMBO J. 1994; 13: 5262-5273Google Scholar).The nucleotide status of Rabs is critical to their function, and it is therefore important to study the nucleotide dependence of Rab geranylgeranylation. This issue has been the subject of previous studies that yielded conflicting results. A study of Rab5 prenylation using a cell-free translation and prenylation assay in rabbit reticulocyte lysates suggested that there was a preference for prenylation of GDP-bound Rab (33Sanford J.C. Pan Y. Wessling-Resnick M. J. Biol. Chem. 1993; 268: 23773-23776Google Scholar). A similar conclusion was recently proposed for Rab6 prenylation in insect cells (34Schiedel A.C. Barnekow A. Mayer T. FEBS Lett. 1995; 376: 113-119Google Scholar). In contrast, in vitro assays using purified or partially purified components suggested that the nucleotide did not influence Rab prenylation (14Seabra M.C. Brown M.S. Slaughter C.A. Sudhof T.C. Goldstein J.L. Cell. 1992; 70: 1049-1057Google Scholar, 35Beranger F. Cadwallader K. Porfiri E. Powers S. Evans T. de Gunzburg J. Hancock J.F. J. Biol. Chem. 1994; 269: 13637-13643Google Scholar).In this study, I analyze the prenylation kinetics of GDP versus GTP-bound forms of Rabs using recombinant purified Rab GGTase and REP and show that there is a clear preference for the GDP-bound form. Furthermore, this effect is shown to be a direct consequence of a higher affinity of REP toward GDP-bound Rab. Inasmuch as Rabs are believed to be GTP-bound after synthesis, these results suggest that a Rab GAP may be involved in presenting newly synthesized Rabs to REP prior to geranylgeranylation.RESULTSIn order to analyze the nucleotide dependence of Rab prenylation, an effective way to load the desired nucleotide after removing all the prebound GDP had to be devised. I adapted a procedure described by Wittinghofer and co-workers (38John J. Sohmen R. Feuerstein J. Linke R. Wittinghofer A. Goody R.S. Biochemistry. 1990; 29: 6058-6065Google Scholar), later modified by Pfeffer and co-workers (39Shapiro A.D. Riederer M.A. Pfeffer S.R. J. Biol. Chem. 1993; 268: 6925-6931Google Scholar) for the loading of Rab proteins. The procedure involves the exchange of pre-bound GDP for GMPPNP in the presence of alkaline phosphatase. Alkaline phosphatase degrades GDP or GTP to guanosine but does not hydrolyze GMPPNP. Rabs will not bind guanosine, and therefore at the end of the exchange reaction, Rabs are efficiently depleted of GDP and loaded with GMPPNP. The latter can later be exchanged with the desired nucleotide owing to its lower affinity for Rabs when compared with GDP or GTP. This procedure was used to load Rabs with GDP, GTP, and the slowly hydrolyzable analog of GTP, GMPPNP. After loading, an aliquot was analyzed for the presence of guanine nucleotides by ion exchange chromatography. Under the conditions used, GDP, GTP, and GMPPNP elute differentially as demonstrated in Fig. 1A. Loading of Rab1a with GDP or GMPPNP resulted in homogenous preparations of GDP-Rab1a (Fig. 1B) and GMPPNP-Rab1a (Fig. 1D), respectively. The GTP-Rab1a preparation contained a small amount of GDP (8%), possibly due to intrinsic hydrolysis during the procedure and some GMPPNP (24%) due to incomplete exchange after the initial exchange of GMPPNP for pre-bound GDP (Fig. 1C).To analyze the effect of the bound nucleotide on the kinetics of Rab prenylation, the loaded Rabs were used as substrates in the in vitro prenylation assay in the presence of recombinant RabGGTase and REP-1. When GDP-loaded Rab1a was used in the assay, an apparent Km of 2.1 µM was obtained (Fig. 2). This result is similar to previously reported Km values obtained with recombinant Rab1a (19Shen F. Seabra M.C. J. Biol. Chem. 1996; 271: 3692-3699Google Scholar, 25Seabra M.C. Ho Y.K. Anant J.S. J. Biol. Chem. 1995; 270: 24420-24427Google Scholar). In contrast, there was a dramatic change in the apparent Km of the reaction when GTP-Rab1a or GMPPNP-Rab1a were used. Because of the low affinity, I was unable to obtain sufficiently concentrated preparations of GTP-Rab1a or GMPPNP-Rab1a to saturate the reaction. However, it is clear that the apparent Km is at least 10-fold higher for GTP-Rab1a and 50-fold for GMPPNP-Rab1a when compared with GDP-Rab1a. To test the generality of this effect, Rab2, Rab3a, and Rab5 were loaded with either GDP and GMPPNP and tested in the in vitro prenylation assay (Fig. 3). In every case, there was a dramatic difference in apparent Km comparable with what was observed in Fig. 2 for Rab1a. The least dramatic change was observed with Rab2, possibly because the GMPPNP-Rab2 preparation was contaminated with 8% of GDP (data not shown).Fig. 3Geranylgeranylation of Rab2, Rab3a, and Rab5: effect of loaded nucleotide. Each reaction mixture contained in a final volume of 50 µl, 5.5 µM [3H]GGPP (3,000 dpm/pmol), 100 ng of RabGGTase, and 100 ng of REP-1, and the indicated concentrations of Rab2 (A), Rab3a (B), or Rab5 (C), loaded with either GDP (closed circles) or GMPPNP (open circles). Duplicate reactions were incubated for 15 min at 37°C, and the amount of [3H]GG transferred to Rab proteins was determined as described under “Experimental Procedures.” A blank value determined in the absence of Rab (0.18 pmol/tube) was subtracted from each point.View Large Image Figure ViewerDownload (PPT)There are two likely explanations for the observed effect on the prenylation kinetics. One is that REP binds with higher affinity to GDP-Rab, and the second is that REP binds equally well to both forms of nucleotide loaded Rab but that only REP·GDP-Rab is recognized by RabGGTase. To begin to discriminate between these two alternative possibilities, a competition experiment was devised using a mutant Rab1a, designated Rab1aSS. Rab1aSS is unable to serve as a prenyl acceptor because the two cysteine residues that accept geranylgeranyl groups are mutated to serine residues. Rab1aSS retains the ability to bind to REP (19Shen F. Seabra M.C. J. Biol. Chem. 1996; 271: 3692-3699Google Scholar), and therefore it is a competitive inhibitor of Rab prenylation (14Seabra M.C. Brown M.S. Slaughter C.A. Sudhof T.C. Goldstein J.L. Cell. 1992; 70: 1049-1057Google Scholar, 19Shen F. Seabra M.C. J. Biol. Chem. 1996; 271: 3692-3699Google Scholar). Susceptibility to Rab1aSS inhibition can serve as an indirect measure of REP-Rab binding. Rab1aSS was loaded with either GDP or GMPPNP and tested as an inhibitor of the prenylation of GDP-Rab1a (Rab1aCC). When increasing amounts of GDP-Rab1aSS were added, we observed up to a 70% reduction in the amount of [3H]GG incorporated into GDP-Rab1a-CC when the mutant protein was present at 30-fold higher concentrations (Fig. 4). At the same high concentration in the assay, GMPPNP-Rab1aSS failed to inhibit the reaction. This result suggests that the GTP-bound Rabs are unable to bind REP with high affinity.Fig. 4Geranylgeranylation of Rab1a in the presence of Rab1a-SS mutant. Each reaction mixture containing in a final volume of 50 µl, 5.5 µM [3H]GGPP (3,000 dpm/pmol), 100 ng of RabGGTase, and 100 ng of REP-1, and 1 µM GDP-loaded Rab1a was incubated at 37°C in the presence of the indicated concentrations of Rab1a-SS loaded with either GDP (closed circles) or GMPPNP (open circles). Duplicate reactions were incubated for 15 min at 37°C, and the amount of [3H]GG transferred to Rab1a proteins was determined as described under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT)To further test the hypothesis that REP has low affinity for GTP-bound Rabs, two distinct direct REP·Rab binding assays were developed. In the first experiment, a gel filtration assay to ascertain REP·Rab complex formation was used (19Shen F. Seabra M.C. J. Biol. Chem. 1996; 271: 3692-3699Google Scholar). When GDP-Rab1a was subjected to gel filtration chromatography on a Superdex 200 column, it eluted as a 25-kDa protein as expected of a monomeric protein and as shown previously (19Shen F. Seabra M.C. J. Biol. Chem. 1996; 271: 3692-3699Google Scholar) (Fig. 5A). When GDP-Rab1a was incubated with REP-1 and the mixture was subjected to gel filtration under the same conditions, a significant fraction of the GDP-Rab1a (approximately 50%) was shifted to fractions 7 and 8, co-eluting with REP-1 (Fig. 5B). In contrast, when GTP-Rab1a was used only a small amount shifted to fractions 7 and 8 (Fig. 5C), and when GMPPNP-Rab1a was used no significant amount of Rab1a was found eluting in fractions 7 and 8 even after long exposures (Fig. 5D). These results confirm the suggestion derived from the previous experiments that REP has higher affinity for GDP-bound Rabs.Fig. 5Detection of REP·Rab1a complex by gel filtration chromatography. Each reaction mixture contained in a final volume of 50 µl, 2 µM of Rab1a-GDP (A and B), Rab1a-GTP (C), or Rab1a-GMPPNP (D), in the absence (Panel A) or the presence of 2 µM REP-1 (B-D). After incubation for 10 min at 37°C, each sample was loaded onto a Superdex 200 3.2/30 column equilibrated and run as described under “Experimental Procedures.” An aliquot (30 µl) of elution fractions 4–12 was subjected to SDS gel electrophoresis on 12.5% mini gels, and the proteins were transferred to nitrocellulose and detected with either J905 anti-REP-1 antibody (0.03 µg/ml) or D576 anti-Rab1a antibody (2.5 µg/ml), as indicated, by immunoblot analysis. Horizontal arrows denote the position of migration of REP and Rab1a (left side), and the molecular mass markers (right side) upon SDS gel electrophoresis are indicated.View Large Image Figure ViewerDownload (PPT)To further study the ability of REP to form a stable complex with GTP-bound Rab, I used a different REP·Rab binding assay (15Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P.M. Goldstein J.L. Cell. 1993; 73: 1091-1099Google Scholar). This REP·Rab binding assay is based on the selective precipitation of his-tagged REP-1 using nickel-Sepharose. If a stable complex between his-tagged REP-1 and non-His-tagged Rab1a is formed, the nickel-Sepharose pellet will contain detectable amounts of guanine nucleotide associated with Rab in the REP·Rab complex. Non-His-tagged Rab1a was loaded with [α-32P]GTP and incubated with REP-1 in the presence or the absence of Rab GGTase. After precipitation of his-tagged REP-1 with nickel-Sepharose, the nucleotide present in the pellet and supernatant was analyzed by thin-layer chromatography. When His-tagged REP-1 was added to the incubation mixture, essentially all of the nucleotide in the nickel-Sepharose pellet was GDP (Fig. 6, lane 2). When His-tagged REP-1 was omitted from the incubation mixture, no nucleotide was present in the pellet (Fig. 6, lane 1). When Rab GGTase was present and prenylation occurred, the nucleotide in the nickel-Sepharose pellet was approximately 75% GDP and 25% GTP (Fig. 6, lane 3). When the supernatant of the reaction was analyzed, the majority of the nucleotide was GTP (80%), as expected because Rab1a was loaded with GTP (Fig. 6, lane 4). This experiment clearly indicates that even in the presence of an excess of GTP-Rab, REP forms a stable complex preferentially with GDP-Rab. A somewhat puzzling finding in this experiment is that the presence of Rab GGTase in the incubation mixture leads to the co-precipitation of some GTP-bound Rab with REP (Fig. 6, lane 3), whereas no GTP-Rab coprecipitates with REP in its absence (Fig. 6, lane 2). There is no obvious explanation for this finding. It is possible that the higher concentration of GTP-Rab in the assay leads to transient complex formation with REP. If the binding of Rab GGTase to the REP·Rab complex is not nucleotide-sensitive, then prenylation will occur and result in the formation of a prenylated GTP-Rab·REP complex. This complex may be sufficiently stable to allow detection in this assay.Fig. 6Detection of Rab-bound nucleotide in the REP·Rab complex by nickel-Sepharose affinity precipitation. Non-His-tagged Rab1a was loaded with [α-32P]GTP and incubated in the presence (+) or absence (−) of His-REP1 and RabGGTase as indicated and described under “Experimental Procedures.” After incubation, the samples were subjected to nickel-Sepharose precipitation, and aliquots of the pellet and supernatant were analyzed by thin-layer chromatography as described under “Experimental Procedures.” Brackets indicate the position of migration of GTP and GDP.View Large Image Figure ViewerDownload (PPT)In the experiment shown in Fig. 6, the REP·Rab complex contained [32P]GDP even though [32P]GTP was the only nucleotide added in the loading reaction. One source of the [32P]GDP was a small contamination of the [32P]GTP stock with [32P]GDP. It is also possible that some GDP-Rab was derived from GTP-Rab by GTP hydrolysis during the loading and incubation procedures. Rabs have slow intrinsic GTPase activity, and it is possible that REP could stimulate their intrinsic GTPase activity, i.e., REP could act as a GAP. To test this hypothesis, REP was incubated with GDP-, GTP-, or GMPPNP-Rab, in the presence of [3H]GGPP and Rab GGTase, and the initial rate of GG transfer was measured. The reaction rate was extremely fast when GDP-Rab was used (Fig. 7, open squares) and slow in the presence of GTP-Rab (Fig. 7, open squares) or GMPPNP-Rab (Fig. 7, open triangles). The difference in the rate of geranylgeranylation of GDP-Rab versus GTP-Rab provided the basis for an assay that measures the formation of GDP-Rab. If GDP-Rab is generated from GTP-Rab, the rate of the reaction will increase. When REP was preincubated with GTP-Rab for 30 min prior to the addition of GGPP and Rab GGTase, the rate of the reaction did not change when compared with the rate obtained in the absence of preincubation (Fig. 7, compare open and closed circles). It is concluded that the REP-Rab preincubation did not result in the formation of GDP-Rab, suggesting that REP has no GAP activity. We also attempted to measure the ability of REP to induce GTP hydrolysis using a standard GAP assay, where Rab1a was loaded with [α-32P]GTP and the hydrolysis of the bound nucleotide was determined by thin-layer chromatography (40Tavitian A. Zahraoui A. Methods Enzymol. 1992; 219: 387-397Google Scholar). Under many different experimental conditions, no significant REP-dependent stimulation of GTP hydrolysis was observed (data not shown).Fig. 7Initial rate of Rab geranylgeranylation: effect of preincubation with REP. Each reaction mixture contained in a final volume of 50 µl, 50 mM sodium Hepes (pH 7.2), 5 mM MgCl2, and 1 mM dithiothreitol, in the presence (closed symbols) or the absence (open symbols) of 2 µM REP-1 and 2 µM of GDP-Rab1a (squares), GTP-Rab1a (circles), or GMPPNP-Rab1a (triangles). After 30 min at 37°C, 50 µl of a standard reaction mixture containing 5.5 µM [3H]GGPP (3,000 dpm/pmol) and 0.5 µM RabGGTase in the presence (open symbols) or the absence (filled symbols) of 2 µM REP-1 was added to each tube and incubated at 37°C. At the indicated times, a 20-µl aliquot from each tube was removed, and the amount of [3H]GG transferred to Rab1a proteins was determined as described under “Experimental Procedures.” A blank value determined in the absence of Rab (open diamonds) was obtained for each time point.View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe current study suggests that REP has a strict preference for GDP-bound Rabs. The formation of a stable REP·GDP-Rab complex initiates digeranylgeranylation by Rab GGTase, followed by membrane translocation of digeranylgeranylated GDP-Rab.As discussed in the introduction, the specificity of the nucleotide requirement has been studied previously with conflicting results. A study of Rab5 prenylation using a cell-free translation and prenylation assay in rabbit reticulocyte lysates suggested a preference for GDP-bound Rab (33Sanford J.C. Pan Y. Wessling-Resnick M. J. Biol. Chem. 1993; 268: 23773-23776Google Scholar). The authors compared the prenylation kinetics of wild type and mutant Rab5 proteins that alter the affinity for guanine nucleotides. Wild type Rab5 was prenylated faster than mutants believed to assume preferentially the GTP-bound state (Rab5Q79L) or the nucleotide-free state (Rab5N133I). More recently, an analysis of Rab6 revealed that Rab6Q72R, a GTPase-deficient mutant equivalent to Rab5Q79L was also less efficiently prenylated upon expression in insect cells (34Schiedel A.C. Barnekow A. Mayer T. FEBS Lett. 1995; 376: 113-119Google Scholar). In contrast, in vitro assays using purified or partially purified components yielded opposite results. In the original study where the purification of Rab GGTase was reported, we found in preliminary experiments that Rab3a prenylation could occur in the presence of either GDP, GTP, or GTP a