VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. (original) (raw)

• Journal List
• EMBO J
• v.13(6); 1994 Mar 15
• PMC394945

EMBO J. 1994 Mar 15; 13(6): 1297–1309.

Division of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla 92093-0668.

Abstract

Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps21, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps21p is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and > 70% similarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps21/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps21p function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 degrees C, (ii) missort multiple vacuolar proteins, (iii) accumulate 40-50 nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kDa protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps21p. We propose that Vps21p functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole.

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• Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed] [Google Scholar]
• Anuntalabhochai S, Terryn N, Van Montagu M, Inzé D. Molecular characterization of an Arabidopsis thaliana cDNA encoding a small GTP-binding protein, Rha1. Plant J. 1991 Sep;1(2):167–174. [PubMed] [Google Scholar]
• Armstrong J, Craighead MW, Watson R, Ponnambalam S, Bowden S. Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator. Mol Biol Cell. 1993 Jun;4(6):583–592. [PMC free article] [PubMed] [Google Scholar]
• Armstrong SA, Seabra MC, Südhof TC, Goldstein JL, Brown MS. cDNA cloning and expression of the alpha and beta subunits of rat Rab geranylgeranyl transferase. J Biol Chem. 1993 Jun 5;268(16):12221–12229. [PubMed] [Google Scholar]
• Balch WE. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 1990 Dec;15(12):473–477. [PubMed] [Google Scholar]
• Bankaitis VA, Johnson LM, Emr SD. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9075–9079. [PMC free article] [PubMed] [Google Scholar]
• Banta LM, Robinson JS, Klionsky DJ, Emr SD. Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J Cell Biol. 1988 Oct;107(4):1369–1383. [PMC free article] [PubMed] [Google Scholar]
• Becker J, Tan TJ, Trepte HH, Gallwitz D. Mutational analysis of the putative effector domain of the GTP-binding Ypt1 protein in yeast suggests specific regulation by a novel GAP activity. EMBO J. 1991 Apr;10(4):785–792. [PMC free article] [PubMed] [Google Scholar]
• Goud B, Salminen A, Walworth NC, Novick PJ. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. [PubMed] [Google Scholar]
• Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992 Sep 4;70(5):715–728. [PubMed] [Google Scholar]
• Calés C, Hancock JF, Marshall CJ, Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. [PubMed] [Google Scholar]
• Chavrier P, Parton RG, Hauri HP, Simons K, Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. [PubMed] [Google Scholar]
• Deng WP, Nickoloff JA. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. [PubMed] [Google Scholar]
• Dieckmann CL, Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
• Dunn SD. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. [PubMed] [Google Scholar]
• Feig LA, Cooper GM. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. [PMC free article] [PubMed] [Google Scholar]
• Glomset JA, Gelb MH, Farnsworth CC. Prenyl proteins in eukaryotic cells: a new type of membrane anchor. Trends Biochem Sci. 1990 Apr;15(4):139–142. [PubMed] [Google Scholar]
• Gorvel JP, Chavrier P, Zerial M, Gruenberg J. rab5 controls early endosome fusion in vitro. Cell. 1991 Mar 8;64(5):915–925. [PubMed] [Google Scholar]
• Goud B, Salminen A, Walworth NC, Novick PJ. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. [PubMed] [Google Scholar]
• Graham TR, Emr SD. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biol. 1991 Jul;114(2):207–218. [PMC free article] [PubMed] [Google Scholar]
• Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed] [Google Scholar]
• Herman PK, Emr SD. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. [PMC free article] [PubMed] [Google Scholar]
• Holcomb CL, Hansen WJ, Etcheverry T, Schekman R. Secretory vesicles externalize the major plasma membrane ATPase in yeast. J Cell Biol. 1988 Mar;106(3):641–648. [PMC free article] [PubMed] [Google Scholar]
• Horazdovsky BF, Emr SD. The VPS16 gene product associates with a sedimentable protein complex and is essential for vacuolar protein sorting in yeast. J Biol Chem. 1993 Mar 5;268(7):4953–4962. [PubMed] [Google Scholar]
• Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed] [Google Scholar]
• Jiang Y, Rossi G, Ferro-Novick S. Bet2p and Mad2p are components of a prenyltransferase that adds geranylgeranyl onto Ypt1p and Sec4p. Nature. 1993 Nov 4;366(6450):84–86. [PubMed] [Google Scholar]
• Johnson LM, Bankaitis VA, Emr SD. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. [PubMed] [Google Scholar]
• Karlin S, Altschul SF. Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2264–2268. [PMC free article] [PubMed] [Google Scholar]
• Kleid DG, Yansura D, Small B, Dowbenko D, Moore DM, Grubman MJ, McKercher PD, Morgan DO, Robertson BH, Bachrach HL. Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science. 1981 Dec 4;214(4525):1125–1129. [PubMed] [Google Scholar]
• Klionsky DJ, Emr SD. Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 1989 Aug;8(8):2241–2250. [PMC free article] [PubMed] [Google Scholar]
• Klionsky DJ, Banta LM, Emr SD. Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Mol Cell Biol. 1988 May;8(5):2105–2116. [PMC free article] [PubMed] [Google Scholar]
• Klionsky DJ, Herman PK, Emr SD. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. [PMC free article] [PubMed] [Google Scholar]
• Kornfeld S. Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors. Annu Rev Biochem. 1992;61:307–330. [PubMed] [Google Scholar]
• Kornfeld S, Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. [PubMed] [Google Scholar]
• McNeil JB, Friesen JD. Expression of the Herpes simplex virus thymidine kinase gene in Saccharomyces cerevisiae. Mol Gen Genet. 1981;184(3):386–393. [PubMed] [Google Scholar]
• Magee T, Newman C. The role of lipid anchors for small G proteins in membrane trafficking. Trends Cell Biol. 1992 Nov;2(11):318–323. [PubMed] [Google Scholar]
• Martinez-Arias AE, Casadaban MJ. Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene. Mol Cell Biol. 1983 Apr;3(4):580–586. [PMC free article] [PubMed] [Google Scholar]
• Novick P, Field C, Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. [PubMed] [Google Scholar]
• Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. [PubMed] [Google Scholar]
• Paravicini G, Horazdovsky BF, Emr SD. Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell. 1992 Apr;3(4):415–427. [PMC free article] [PubMed] [Google Scholar]
• Pfeffer SR. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. [PubMed] [Google Scholar]
• Pfeffer SR, Rothman JE. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. [PubMed] [Google Scholar]
• Powers S. Protein prenylation: a modification that sticks. Curr Biol. 1991 Apr;1(2):114–116. [PubMed] [Google Scholar]
• Pringle JR, Preston RA, Adams AE, Stearns T, Drubin DG, Haarer BK, Jones EW. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. [PubMed] [Google Scholar]
• Pryer NK, Wuestehube LJ, Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. [PubMed] [Google Scholar]
• Raths S, Rohrer J, Crausaz F, Riezman H. end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993 Jan;120(1):55–65. [PMC free article] [PubMed] [Google Scholar]
• Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell. 1992 Dec;3(12):1389–1402. [PMC free article] [PubMed] [Google Scholar]
• Redding K, Holcomb C, Fuller RS. Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae. J Cell Biol. 1991 May;113(3):527–538. [PMC free article] [PubMed] [Google Scholar]
• Riezman H. Yeast endocytosis. Trends Cell Biol. 1993 Aug;3(8):273–277. [PubMed] [Google Scholar]
• Robinson JS, Klionsky DJ, Banta LM, Emr SD. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988 Nov;8(11):4936–4948. [PMC free article] [PubMed] [Google Scholar]
• Rossi G, Yu JA, Newman AP, Ferro-Novick S. Dependence of Ypt1 and Sec4 membrane attachment on Bet2. Nature. 1991 May 9;351(6322):158–161. [PubMed] [Google Scholar]
• Rothman JH, Stevens TH. Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell. 1986 Dec 26;47(6):1041–1051. [PubMed] [Google Scholar]
• Rothman JH, Howald I, Stevens TH. Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae. EMBO J. 1989 Jul;8(7):2057–2065. [PMC free article] [PubMed] [Google Scholar]
• Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed] [Google Scholar]
• Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. [PubMed] [Google Scholar]
• Schimmöller F, Riezman H. Involvement of Ypt7p, a small GTPase, in traffic from late endosome to the vacuole in yeast. J Cell Sci. 1993 Nov;106(Pt 3):823–830. [PubMed] [Google Scholar]
• Schwaninger R, Plutner H, Bokoch GM, Balch WE. Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes. J Cell Biol. 1992 Dec;119(5):1077–1096. [PMC free article] [PubMed] [Google Scholar]
• Segev N, Mulholland J, Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. [PubMed] [Google Scholar]
• Serrano R. Structure, function and regulation of plasma membrane H(+)-ATPase. FEBS Lett. 1993 Jun 28;325(1-2):108–111. [PubMed] [Google Scholar]
• Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PMC free article] [PubMed] [Google Scholar]
• Singer B, Riezman H. Detection of an intermediate compartment involved in transport of alpha-factor from the plasma membrane to the vacuole in yeast. J Cell Biol. 1990 Jun;110(6):1911–1922. [PMC free article] [PubMed] [Google Scholar]
• Stevens T, Esmon B, Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. [PubMed] [Google Scholar]
• Tan PK, Davis NG, Sprague GF, Payne GS. Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast. J Cell Biol. 1993 Dec;123(6 Pt 2):1707–1716. [PMC free article] [PubMed] [Google Scholar]
• Tisdale EJ, Bourne JR, Khosravi-Far R, Der CJ, Balch WE. GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol. 1992 Nov;119(4):749–761. [PMC free article] [PubMed] [Google Scholar]
• Vida TA, Huyer G, Emr SD. Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment. J Cell Biol. 1993 Jun;121(6):1245–1256. [PMC free article] [PubMed] [Google Scholar]
• Walworth NC, Goud B, Ruohola H, Novick PJ. Fractionation of yeast organelles. Methods Cell Biol. 1989;31:335–356. [PubMed] [Google Scholar]
• Walworth NC, Brennwald P, Kabcenell AK, Garrett M, Novick P. Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2017–2028. [PMC free article] [PubMed] [Google Scholar]
• Wichmann H, Hengst L, Gallwitz D. Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p). Cell. 1992 Dec 24;71(7):1131–1142. [PubMed] [Google Scholar]
• Wilsbach K, Payne GS. Vps1p, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. EMBO J. 1993 Aug;12(8):3049–3059. [PMC free article] [PubMed] [Google Scholar]
• Wilson AL, Maltese WA. Isoprenylation of Rab1B is impaired by mutations in its effector domain. J Biol Chem. 1993 Jul 15;268(20):14561–14564. [PubMed] [Google Scholar]
• Wright R, Basson M, D'Ari L, Rine J. Increased amounts of HMG-CoA reductase induce "karmellae": a proliferation of stacked membrane pairs surrounding the yeast nucleus. J Cell Biol. 1988 Jul;107(1):101–114. [PMC free article] [PubMed] [Google Scholar]
• Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed] [Google Scholar]

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