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• W2040572812 abstract "A yeast open reading frame sharing homology with the two known yeast Rab GTPase-activating proteins (GAPs), Gyp6p and Gyp7p, was found in a data base search. We have named the gene containing this open reading frame GYP1. Recombinant Gyp1p showed GAP activity on Sec4p, increasing both its steady-state rate and single turnover GTPase activity. Gyp1p also stimulated the GTPase activity of several other yeast Rab proteins including Ypt1p, Ypt7p, and Ypt51p but showed no GAP activity on Ypt6p and Ypt32p. Deletion of the GYP1 gene or overexpression of Gyp1p did not alter the growth rate of yeast. However, overexpression of Gyp1p was inhibitory in combination with a subset of secretory mutants includingsec4-8 and several ypt1 mutants. This effect is probably due to the increase in GAP activity, which can be observed in a lysate from cells overexpressing Gyp1p. The finding that yeast Rab GAPs share homology with proteins in other species, such asCaenorhabditis elegans and human, suggests the existence of a conserved Rab GAP family. A yeast open reading frame sharing homology with the two known yeast Rab GTPase-activating proteins (GAPs), Gyp6p and Gyp7p, was found in a data base search. We have named the gene containing this open reading frame GYP1. Recombinant Gyp1p showed GAP activity on Sec4p, increasing both its steady-state rate and single turnover GTPase activity. Gyp1p also stimulated the GTPase activity of several other yeast Rab proteins including Ypt1p, Ypt7p, and Ypt51p but showed no GAP activity on Ypt6p and Ypt32p. Deletion of the GYP1 gene or overexpression of Gyp1p did not alter the growth rate of yeast. However, overexpression of Gyp1p was inhibitory in combination with a subset of secretory mutants includingsec4-8 and several ypt1 mutants. This effect is probably due to the increase in GAP activity, which can be observed in a lysate from cells overexpressing Gyp1p. The finding that yeast Rab GAPs share homology with proteins in other species, such asCaenorhabditis elegans and human, suggests the existence of a conserved Rab GAP family. GTP binding proteins cycle between the GTP-bound active form and the GDP-bound inactive form. The interconversion between these two forms depends on the rates of guanine nucleotide exchange and GTP hydrolysis. Both of these reactions are intrinsically slow. But in the cell, they are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), 1The abbreviations used are: GAP, GTPase-activating protein; ORF, open reading frame; GTPγS, guanosine 5′-3-O-(thio)triphosphate. respectively (1Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1761) Google Scholar). Rab proteins constitute the largest family of small GTP-binding proteins (2Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (664) Google Scholar). Eleven genes encoding Rab proteins (SEC4, YPT1, YPT31, YPT32, YPT51, YPT52, YPT53, YPT6, YPT7, YBR264C, and YNL304W) are found in yeast where they play important roles in vesicle transport. For example, Sec4p, the first Rab protein to be implicated in vesicle traffic, is involved in the last stage of the secretory pathway in yeast (3Salminen A. Novick P.J. Cell. 1987; 49: 527-538Abstract Full Text PDF PubMed Scopus (593) Google Scholar). When the function of Sec4p is blocked, cells accumulate Golgi-derived secretory vesicles. GAP activities for Rab proteins have been observed for a long time (4Tan T.J. Vollmer P. Gallwitz D. FEBS Lett. 1991; 291: 322-326Crossref PubMed Scopus (20) Google Scholar, 5Burstein E.S. Linko-Stentz K. Lu Z.J. Macara I.G. J. Biol. Chem. 1991; 266: 2689-2692Abstract Full Text PDF PubMed Google Scholar, 6Walworth N.C. Brennwald P. Kabcenell A.K. Garrett M. Novick P. Mol. Cell. Biol. 1992; 12: 2017-2028Crossref PubMed Scopus (108) Google Scholar). A yeast protein called Gyp6p was the first Rab GAP to be cloned (7Strom M. Vollmer P. Tan T.J. Gallwitz D. Nature. 1993; 361: 736-739Crossref PubMed Scopus (146) Google Scholar). It specifically activates the GTPase activity of Ypt6p and Ypt7p. To date, the only other Rab GAP that has been identified in yeast is Gyp7p (8Vollmer P. Gallwitz D. Methods Enzymol. 1995; 257: 118-128Crossref PubMed Scopus (26) Google Scholar), which is also active on Ypt7p. Recently, a mammalian GAP specific for the Rab3 subfamily was purified and cloned (9Fukui K. Sasaki T. Imazumi K. Matsuura Y. Nakanishi H. Takai Y. J. Biol. Chem. 1997; 272: 4655-4658Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). This protein has no homology to the yeast GAPs and appears to have no homolog in the yeast genome. A Sec4p-specific GAP activity in the membrane fraction of a yeast lysate was previously observed (6Walworth N.C. Brennwald P. Kabcenell A.K. Garrett M. Novick P. Mol. Cell. Biol. 1992; 12: 2017-2028Crossref PubMed Scopus (108) Google Scholar). To facilitate cloning the Sec4p-specific GAP, we searched the sequence data base and found an uncharacterized yeast ORF that shares homology with Gyp6p, Gyp7p, and a family of proteins from other species. In this paper, we demonstrate that this protein is a GAP for Sec4p. The construct for expressing His6-tagged Sec4p was described in Ref. 10Collins R.N. Brennwald P. Garrett M. Lauring A. Novick P. J. Biol. Chem. 1997; 272: 18281-18289Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar. The coding regions of other yeast Rab proteins used in this study and Gyp1p were synthesized by polymerase chain reaction and inserted into pET15b (Novagen, Inc., Madison, WI), so that they were all tagged with six histidine residues at their N termini. Recombinant fusion proteins were induced for 2 h at 37 °C with 0.4 mm isopropyl-β-d-thiogalactopyranoside, except that Gyp1p was induced for 4 h at 25 °C. Purification was performed with Ni2+-nitrilotriacetic acid-agarose (Qiagen, Inc., Chatsworth, CA), following the protocol suggested by the manufacturer. All of the purified Rab proteins were more than 90% pure, as judged by Coomassie Brilliant Blue staining of sodium dodecyl sulfate polyacrylamide gels. Because Gyp1p was degraded in the cell or during purification, some truncated forms ranging from 60 to 70 kDa co-purified with the 70-kDa full-length protein. Guanine nucleotide analysis and charcoal binding method were performed as described previously (6Walworth N.C. Brennwald P. Kabcenell A.K. Garrett M. Novick P. Mol. Cell. Biol. 1992; 12: 2017-2028Crossref PubMed Scopus (108) Google Scholar). All experiments were carried out at room temperature. The concentrations of Rab proteins were defined by their [35S]GTPγS binding capacity. The concentration of recombinant Gyp1p was determined by Bradford method using bovine serum albumin as standard. For the analysis of nucleotide product, [α-32P]GTP was incubated for 20 min with Sec4p in buffer A (50 mm Na-HEPES, pH 6.8, 1 mm ATP, 1 mg/ml bovine serum albumin, 1 mm dithiothreitol) containing 5 mm MgCl2. Gyp1p in the same buffer was added at zero time. Samples were taken at 0 and 30 min, and the nucleotides were resolved by thin layer chromatography. The steady-state rates were measured by directly adding Sec4p into the reaction mix containing buffer A, 5 mm MgCl2and excess [γ-32P]GTP. For the measurement of single turnover rate, Sec4p and Ypt6p were preloaded with [γ-32P]GTP in buffer A containing 5 mmMgCl2; Ypt1p, Ypt7p, Ypt32p, and Ypt51p were preloaded in buffer A containing 1 mm EDTA, 0.5 mmMgCl2. The assay mix contained Buffer A, 5 mmMgCl2 and 1 mm unlabeled GTP. We adopted a method similar to the so called Intergenic Flip Flop (11Mallet L. Jacquet M. Yeast. 1996; 12: 1351-1357Crossref PubMed Scopus (16) Google Scholar) to produce in one experiment both a disrupting cassette and a plasmid for gap repair. Two regions upstream and downstream of the Gyp1p open reading frame were polymerase chain reaction amplified from genomic DNA. They were either inserted into a centromere-based plasmid in the same orientation as in genomic DNA for the cloning of GYP1 by gap repair or inserted into an integrating plasmid in the opposite orientation for the disruption of GYP1. Transformations were performed by the lithium acetate method (12Gietz R.D. Schiestl R.H. Methods Mol. Cell. Biol. 1995; 5: 255-269Google Scholar). The integration of the disrupting cassette was confirmed by polymerase chain reaction. The GYP1 gene rescued by gap repair was subcloned into 2μ plasmid for the overexpression of Gyp1p in yeast. Cells were grown overnight to log phase in minimal media plus 1% casamino acid (for yeast transformed with 2μ GYP1 plasmid, and for gyp1deletion mutant), or in minimum media plus 1% casamino acid and uracil (for wild type yeast). After washing once with 10 mmNaN3, the cells were resuspended in ice-cold lysis buffer (100 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, 1 mm dithiothreitol, 5% glycerol, 1 mmbenzamidine, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin, 2 μg/ml leupeptin). Equal volumes of glass beads were added, and cells were broken by vortexing 5 × 1 min at 4 °C, with a 1-min interval in between on ice. Lysates were centrifuged at 450 × g for 3 min. Supernatants were used in the GAP activity measurement. The protein concentration of lysate was determined by the Bradford method and adjusted to 12 mg/ml. 30 μl of lysate or lysis buffer were added to 170 μl of reaction mix containing 50 mm Tris-HCl, pH 8.0, 10 mmMgCl2, 1 mm ATP, 1 mm GTP, 1 mg/ml bovine serum albumin, 1 mm dithiothreitol, and 0.1 μm Rab proteins preloaded with [γ-32P]GTP. Samples were taken at 0, 30, 60, and 90 min and diluted into ice-cold stop buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 5 mm MgCl2). [γ-32P]GTP remaining Rab-associated was determined by the filter binding method (13Kabcenell A.K. Goud B. Northup J.K. Novick P.J. J. Biol. Chem. 1990; 265: 9366-9372Abstract Full Text PDF PubMed Google Scholar). A computer homology search revealed that the two known yeast Rab GAPs, Gyp6p and Gyp7p, share limited homology in two regions. BLAST data base search (14Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70762) Google Scholar) with the peptide sequences from these two regions demonstrated that many other proteins share sequence homology in these two motifs (Fig. 1 A). Among them is an unknown yeast ORF, YOR070C. Its predicted protein sequence consists of 637 amino acid residues with a calculated molecular mass of 73.3 kDa and a predicted isoelectric point of 6.98. We termed this protein Gyp1p, because it possesses GAP activity for Ypt1p and Sec4p. A C. elegans ORF F32B6.h is probably the homolog of Gyp1p in this organism. The two motifs used in the BLAST search are conserved perfectly between Gyp1p and F32B6.h (Fig. 1 B). We also found some human expressed sequence tags that showed very strong homology to Gyp1p and F32B6.h. The homology shared among them is not confined to the two motifs. For example, a stretch of amino acids close to the C-terminal end of Gyp1p matched very well to the sequences in theC. elegans and human homologs (Fig. 1 B). Gyp1p was prepared as a His6-tagged protein fromEscherichia coli. Thin layer chromatography analysis of the GTPase reaction product showed that Gyp1p stimulates the hydrolysis of Sec4p-bound [α-32P]GTP to GDP, but Gyp1p itself does not have GTPase activity (Fig. 2 A). Because of its low intrinsic GTPase activity, Sec4p cycles between the GTP-bound and GDP-bound states very slowly. Therefore, the steady-state rate of GTP hydrolysis by Sec4p alone was almost unmeasurable by the charcoal binding method (Fig. 2 B). Addition of Gyp1p stimulated the rate dramatically, suggesting that the hydrolysis step is a rate-limiting step in the cycle. This is consistent with the fact that Sec4p has a relatively fast intrinsic exchange rate (13Kabcenell A.K. Goud B. Northup J.K. Novick P.J. J. Biol. Chem. 1990; 265: 9366-9372Abstract Full Text PDF PubMed Google Scholar). However, the highest steady-state rate in this experiment was achieved by the addition of both Gyp1p and Sec2-59p, the guanine nucleotide exchange factor for Sec4p (15Walch-Solimena C. Collins R.N. Novick P.J. J. Cell. Biol. 1997; 137: 1495-1509Crossref PubMed Scopus (270) Google Scholar), indicating that Sec4p undergoes the GTP-binding/GTPase cycle most efficiently in the presence of both its GAP and guanine nucleotide exchange factors. Under single turnover conditions, concentrations of Gyp1p as low as 1.2 ng/μl, which we estimate to represent a maximal molar concentration of 17 nm, were sufficient to enhance the GTPase activity of 1.4 μm GTP-Sec4p (Fig. 2 C), and the initial slopes of the curves in Fig. 2 C are directly proportional to the Gyp1p concentration, clearly indicating the catalytic nature of the interaction. The maximal enhancement of the rate of GTP single-round hydrolysis in this experiment was greater than 200-fold. Gyp6p was shown to be active on Ypt6p and Ypt7p but not on Sec4p or Ypt1p (7Strom M. Vollmer P. Tan T.J. Gallwitz D. Nature. 1993; 361: 736-739Crossref PubMed Scopus (146) Google Scholar). To test the substrate specificity of Gyp1p, we prepared the recombinant proteins that covered most of the characterized yeast Rab proteins or subfamilies, including Sec4p, Ypt1p, Ypt6p, Ypt7p, Ypt32p, and Ypt51p. The Sec4p GTPase-deficient mutant Sec4L79p was also included in this analysis. As shown in Table I, the GTPase activities of Sec4p, Ypt1p, Ypt7p, Ypt51p, and Sec4L79p were strongly activated by Gyp1p. The same amount of Gyp1p has no significant effect on the GTPase activities of Ypt6p and Ypt32p.Table ISubstrate specificity of Gyp1pGTPaseGTPase activity (fmol/min)Activation ratio−Gyp1p+Gyp1pSec4p9.4086291.7Ypt1p16.195459.2Ypt6p2.424.631.92Ypt7p13.873153.0Ypt32p19.718.10.919Ypt51p25.954120.9Sec4L79p0.88758.966.4 Open table in a new tab To assess the functional significance of Gyp1p, the gene encoding Gyp1p was deleted by replacing the coding region with URA3. Thegyp1 deletion mutant cells grow normally at 25, 16, or 37 °C. Overexpression of Gyp1p from a high copy plasmid has no effect on the growth of wild type yeast cells either. However, when the high copy plasmid containing GYP1 was transformed into secretory mutants, the restrictive temperatures of some mutants were lowered (Table II), indicating a synthetic inhibitory effect. As shown in Table II, the growth ofsec4-8 and all three ypt1 mutants was inhibited by the overexpression of Gyp1p. Most of the other mutants whose growth were inhibited have functional and/or genetic interaction withSEC4 or YPT1. For example, Sec2p is the exchange factor for Sec4p; Bet2p is one subunit of the prenyl transferase essential for the membrane attachment of Sec4p and Ypt1p.Table IIGrowth properties of mutants transformed with 2μ vector or plasmid containing GYP12μ vector control2μGYP1Inhibitory effect25 °C30 °C34 °C37 °C25 °C30 °C34 °C37 °Cypt1–2++++++±−Yesypt1–3+++±++±−Yesypt1ts+++−++−−Yesbet2–1+++±++±−Yessec12–4++−−++−−sec13–1++−−++−−sec16–2+−−−+−−−sec20–1+±−−+±−−sec21–1++−−+±−−Yessec22–3+±−−+±−−sec23–1++−−+±−−Yessec7–1++−−++−−sec14–3+++−++±−Yessec1–1++−−++−−sec2–41++−−+±−−Yessec4–8+±−−±−−−Yessec3–2+++−+++−sec6–4++−−++−−sec8–9+++−+++−sec10–2+++−+++−sec15–1+++−++−−Yessec9–4++−−++−−sec17–1++±−++±−sec18–1+−−−+−−−sec19–1++−−++−− Open table in a new tab Lysates were prepared from wild type yeast, from yeast transformed with the 2μ GYP1 plasmid, and from the gyp1 deletion mutant. The GAP activity for Sec4p, Ypt1p, and Ypt6p in the lysate was measured by the filter binding method (Fig. 3). Compared with the wild type yeast lysate, the lysate from yeast transformed with 2μ GYP1plasmid was more active on Sec4p and Ypt1p but not on Ypt6p, indicating that in vivo overexpression of Gyp1p increases the GAP activity on Sec4p and Ypt1p. This result is consistent with the substrate specificity data obtained with recombinant Gyp1p and suggests that the inhibitory effect of the 2μ GYP1 plasmid on secretory mutants is likely to be caused by the increase of GAP activity. The lysate from the gyp1 deletion mutant showed only a moderate decrease of GAP activity for Sec4p and Ypt1p, implying that Gyp1p is probably not the only GAP for Sec4p and Ypt1p in yeast and is not the major source of the observed GAP activity in a wild type yeast lysate. We demonstrate here that a yeast protein that shares two conserved motifs with a family of proteins, including the two known yeast Rab GAPs, Gyp6p and Gyp7p, is a GAP that acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p. The homology shared by the members of this protein family is not extensive but rather confined to the two motifs, which may be the reason why the homology between Gyp6p and Gyp7p was not previously recognized. The fact that Gyp1p was identified by this homology and proved to be a Rab GAP leads us to hypothesize that all the members of this protein family are likely to have a common biological function, i.e. they have GAP activity for Rab proteins. Furthermore, it is tempting to speculate that the two conserved motifs are parts of the catalytic domain, because GAPs for the Ras and Rho family members share the common catalytic domains specific for each family (1Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1761) Google Scholar). This possibility is being explored experimentally. It is of interest to mention that two arginine residues in the second motif are conserved among all members of this protein family, in light of the finding that arginine residues play important roles in the catalytic activity of Ras and Rho GAPs (16Rittinger K. Walker P.A. Eccleston J.F. Nurmahomed K. Owen D. Laue E. Gamblin S.J. Smerdon S.J. Nature. 1997; 388: 693-697Crossref PubMed Scopus (223) Google Scholar, 17Scheffzek K. Ahmadian M.R. Kabsch W. Wiesm uuml, ller, L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1202) Google Scholar). In the yeast genome, Gyp1p is the only ORF that we found to share significant homology with Gyp6p and Gyp7p. During the preparation of this manuscript, a more extensive sequence similarity between Gyp6p, Gyp7p, and many other proteins including cell cycle proteins Bub2p and Cdc16p has been described, using a less stringent searching algorithm (18Neuwald A.F. Trends Biochem. Sci. 1997; 22: 243-244Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Many residues conserved in the protein family we defined here, such as the two arginine residues mentioned above, are not conserved in those more distantly related proteins. However, the weak homology shared between Rab GAPs and cell cycle checkpoint proteins may have some functional implication. Because the intrinsic GTPase activity of Rab proteins is very low, GAPs are necessary to convert them from their GTP-bound forms to their GDP-bound forms. It is not clear at which point in the functional cycle of Rab proteins the bound GTP is hydrolyzed. Studies on Rab3a have shown that hydrolysis occurs during exocytosis (19Stahl B. von Mollard G.F. Walch-Solimena C. Jahn R. J. Biol. Chem. 1994; 269: 24770-24776Abstract Full Text PDF PubMed Google Scholar). Studies using an XTP-binding mutant of Rab5 have suggested that Rab5 continually cycles between GTP- and GDP-bound forms on the vesicle (20Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M.C. Goody R. Zerial M. Nature. 1996; 383: 266-269Crossref PubMed Scopus (267) Google Scholar). Regardless of where hydrolysis normally occurs in the cell, overexpression of GAPs may down-regulate the level of activated Rab proteins, as is evidenced by our genetic data demonstrating inhibitory effects of Gyp1p overexpression. In addition, Rab GAPs have been hypothesized to aid the process of Rab prenylation, due to the preference of Rab escort protein for the GDP-bound form of Rab proteins. However, the phenotype ofgyp1 deletion mutant does not suggest such a role. Because GAPs may be needed at more than one stage of the Rab protein functional cycle, it is conceivable that in the cell there are several GAPs that may act on a single Rab proteins. That Gyp1p, the only known GAP for the essential yeast Rab proteins Sec4p and Ypt1p, is not necessary for the growth of yeast may imply that there are yet unknown GAPs that play redundant roles or that Gyp1p performs a function not required in the normal growth phase. Our results showing that overexpression of Gyp1p inhibits the growth of sec4, ypt1, and some other secretory mutants support the idea that Rab GAPs negatively regulate the function of Rab proteins and decrease the efficiency of vesicular transport. Our lab has previously identified a Sec4p-specific GAP activity in the membrane fraction of yeast lysate (6Walworth N.C. Brennwald P. Kabcenell A.K. Garrett M. Novick P. Mol. Cell. Biol. 1992; 12: 2017-2028Crossref PubMed Scopus (108) Google Scholar). A competition assay showed that the activity can be specifically inhibited by excess Sec4p but not Ypt1p, implying specificity for Sec4p. Because Gyp1p is active on both Sec4p and Ypt1p, it probably does not account for the membrane-associated Sec4p GAP activity. Purification and cloning of other Sec4p-specific GAPs should enhance our understanding of the functional mechanism of Rab proteins." @default.
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• W2040572812 title "Identification of a Sec4p GTPase-activating Protein (GAP) as a Novel Member of a Rab GAP Family" @default.
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