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EMBO J. Apr 15, 1998; 17(8): 2156–2165.
PMCID: PMC1170560

Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.

Abstract

ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy. COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p. LMA1 and Sec18p are required for vesicle fusion after Uso1p function. Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p. Once docked, however, vesicle fusion is no longer sensitive to GDI. In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding to membranes. To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed. These mutations do not interfere with Uso1p-mediated docking, but block membrane fusion. We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Araki S, Kikuchi A, Hata Y, Isomura M, Takai Y. Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J Biol Chem. 1990 Aug 5;265(22):13007–13015. [PubMed]
  • Baker D, Hicke L, Rexach M, Schleyer M, Schekman R. Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell. 1988 Jul 29;54(3):335–344. [PubMed]
  • Baker D, Wuestehube L, Schekman R, Botstein D, Segev N. GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction. Proc Natl Acad Sci U S A. 1990 Jan;87(1):355–359. [PMC free article] [PubMed]
  • Ballou CE. A study of the immunochemistry of three yeast mannans. J Biol Chem. 1970 Mar 10;245(5):1197–1203. [PubMed]
  • Banfield DK, Lewis MJ, Pelham HR. A SNARE-like protein required for traffic through the Golgi complex. Nature. 1995 Jun 29;375(6534):806–809. [PubMed]
  • Barlowe C. Coupled ER to Golgi transport reconstituted with purified cytosolic proteins. J Cell Biol. 1997 Dec 1;139(5):1097–1108. [PMC free article] [PubMed]
  • Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N, Rexach MF, Ravazzola M, Amherdt M, Schekman R. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994 Jun 17;77(6):895–907. [PubMed]
  • Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. [PubMed]
  • Dascher C, Balch WE. Mammalian Sly1 regulates syntaxin 5 function in endoplasmic reticulum to Golgi transport. J Biol Chem. 1996 Jul 5;271(27):15866–15869. [PubMed]
  • Dascher C, Ossig R, Gallwitz D, Schmitt HD. Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily. Mol Cell Biol. 1991 Feb;11(2):872–885. [PMC free article] [PubMed]
  • Dirac-Svejstrup AB, Soldati T, Shapiro AD, Pfeffer SR. Rab-GDI presents functional Rab9 to the intracellular transport machinery and contributes selectivity to Rab9 membrane recruitment. J Biol Chem. 1994 Jun 3;269(22):15427–15430. [PubMed]
  • Evan GI, Lewis GK, Ramsay G, Bishop JM. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. [PMC free article] [PubMed]
  • Garrett MD, Kabcenell AK, Zahner JE, Kaibuchi K, Sasaki T, Takai Y, Cheney CM, Novick PJ. Interaction of Sec4 with GDI proteins from bovine brain, Drosophila melanogaster and Saccharomyces cerevisiae. Conservation of GDI membrane dissociation activity. FEBS Lett. 1993 Oct 4;331(3):233–238. [PubMed]
  • Garrett MD, Zahner JE, Cheney CM, Novick PJ. GDI1 encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway. EMBO J. 1994 Apr 1;13(7):1718–1728. [PMC free article] [PubMed]
  • Haas A, Scheglmann D, Lazar T, Gallwitz D, Wickner W. The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance. EMBO J. 1995 Nov 1;14(21):5258–5270. [PMC free article] [PubMed]
  • Hardwick KG, Pelham HR. SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex. J Cell Biol. 1992 Nov;119(3):513–521. [PMC free article] [PubMed]
  • Hunt JM, Bommert K, Charlton MP, Kistner A, Habermann E, Augustine GJ, Betz H. A post-docking role for synaptobrevin in synaptic vesicle fusion. Neuron. 1994 Jun;12(6):1269–1279. [PubMed]
  • Kaiser CA, Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 1990 May 18;61(4):723–733. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lian JP, Ferro-Novick S. Bos1p, an integral membrane protein of the endoplasmic reticulum to Golgi transport vesicles, is required for their fusion competence. Cell. 1993 May 21;73(4):735–745. [PubMed]
  • Lian JP, Stone S, Jiang Y, Lyons P, Ferro-Novick S. Ypt1p implicated in v-SNARE activation. Nature. 1994 Dec 15;372(6507):698–701. [PubMed]
  • Lupashin VV, Waters MG. t-SNARE activation through transient interaction with a rab-like guanosine triphosphatase. Science. 1997 May 23;276(5316):1255–1258. [PubMed]
  • Lupashin VV, Hamamoto S, Schekman RW. Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes. J Cell Biol. 1996 Feb;132(3):277–289. [PMC free article] [PubMed]
  • Mayer A, Wickner W, Haas A. Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell. 1996 Apr 5;85(1):83–94. [PubMed]
  • Mizuta K, Warner JR. Continued functioning of the secretory pathway is essential for ribosome synthesis. Mol Cell Biol. 1994 Apr;14(4):2493–2502. [PMC free article] [PubMed]
  • Newman AP, Shim J, Ferro-Novick S. BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex. Mol Cell Biol. 1990 Jul;10(7):3405–3414. [PMC free article] [PubMed]
  • Newman AP, Groesch ME, Ferro-Novick S. Bos1p, a membrane protein required for ER to Golgi transport in yeast, co-purifies with the carrier vesicles and with Bet1p and the ER membrane. EMBO J. 1992 Oct;11(10):3609–3617. [PMC free article] [PubMed]
  • 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]
  • Peter F, Nuoffer C, Pind SN, Balch WE. Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack. J Cell Biol. 1994 Sep;126(6):1393–1406. [PMC free article] [PubMed]
  • Pevsner J, Hsu SC, Braun JE, Calakos N, Ting AE, Bennett MK, Scheller RH. Specificity and regulation of a synaptic vesicle docking complex. Neuron. 1994 Aug;13(2):353–361. [PubMed]
  • Pfeffer SR. Transport vesicle docking: SNAREs and associates. Annu Rev Cell Dev Biol. 1996;12:441–461. [PubMed]
  • Rexach MF, Schekman RW. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol. 1991 Jul;114(2):219–229. [PMC free article] [PubMed]
  • Rexach MF, Latterich M, Schekman RW. Characteristics of endoplasmic reticulum-derived transport vesicles. J Cell Biol. 1994 Sep;126(5):1133–1148. [PMC free article] [PubMed]
  • Richardson CJ, Jones S, Litt RJ, Segev N. GTP hydrolysis is not important for Ypt1 GTPase function in vesicular transport. Mol Cell Biol. 1998 Feb;18(2):827–838. [PMC free article] [PubMed]
  • Rothman JE. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. [PubMed]
  • Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. [PubMed]
  • Rybin V, Ullrich O, Rubino M, Alexandrov K, Simon I, Seabra MC, Goody R, Zerial M. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion. Nature. 1996 Sep 19;383(6597):266–269. [PubMed]
  • Salama NR, Yeung T, Schekman RW. The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins. EMBO J. 1993 Nov;12(11):4073–4082. [PMC free article] [PubMed]
  • Sapperstein SK, Walter DM, Grosvenor AR, Heuser JE, Waters MG. p115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Golgi transport factor Uso1p. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):522–526. [PMC free article] [PubMed]
  • Sapperstein SK, Lupashin VV, Schmitt HD, Waters MG. Assembly of the ER to Golgi SNARE complex requires Uso1p. J Cell Biol. 1996 Mar;132(5):755–767. [PMC free article] [PubMed]
  • Sasaki T, Kikuchi A, Araki S, Hata Y, Isomura M, Kuroda S, Takai Y. Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein. J Biol Chem. 1990 Feb 5;265(4):2333–2337. [PubMed]
  • Schekman R, Orci L. Coat proteins and vesicle budding. Science. 1996 Mar 15;271(5255):1526–1533. [PubMed]
  • Schröder S, Schimmöller F, Singer-Krüger B, Riezman H. The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP. J Cell Biol. 1995 Nov;131(4):895–912. [PMC free article] [PubMed]
  • 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]
  • Segev N. Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein. Science. 1991 Jun 14;252(5012):1553–1556. [PubMed]
  • Seog DH, Kito M, Igarashi K, Yoda K, Yamasaki M. Molecular characterization of the USO1 gene product which is essential for vesicular transport in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1994 Apr 15;200(1):647–653. [PubMed]
  • Shim J, Newman AP, Ferro-Novick S. The BOS1 gene encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast. J Cell Biol. 1991 Apr;113(1):55–64. [PMC free article] [PubMed]
  • 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]
  • Søgaard M, Tani K, Ye RR, Geromanos S, Tempst P, Kirchhausen T, Rothman JE, Söllner T. A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell. 1994 Sep 23;78(6):937–948. [PubMed]
  • Söllner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. [PubMed]
  • Stenmark H, Vitale G, Ullrich O, Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell. 1995 Nov 3;83(3):423–432. [PubMed]
  • Stone S, Sacher M, Mao Y, Carr C, Lyons P, Quinn AM, Ferro-Novick S. Bet1p activates the v-SNARE Bos1p. Mol Biol Cell. 1997 Jul;8(7):1175–1181. [PMC free article] [PubMed]
  • Südhof TC. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995 Jun 22;375(6533):645–653. [PubMed]
  • TerBush DR, Novick P. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J Cell Biol. 1995 Jul;130(2):299–312. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Ullrich O, Stenmark H, Alexandrov K, Huber LA, Kaibuchi K, Sasaki T, Takai Y, Zerial M. Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins. J Biol Chem. 1993 Aug 25;268(24):18143–18150. [PubMed]
  • Ungermann C, Nichols BJ, Pelham HR, Wickner W. A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion. J Cell Biol. 1998 Jan 12;140(1):61–69. [PMC free article] [PubMed]
  • Walch-Solimena C, Blasi J, Edelmann L, Chapman ER, von Mollard GF, Jahn R. The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling. J Cell Biol. 1995 Feb;128(4):637–645. [PMC free article] [PubMed]
  • Wuestehube LJ, Duden R, Eun A, Hamamoto S, Korn P, Ram R, Schekman R. New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the endoplasmic reticulum to the Golgi complex. Genetics. 1996 Feb;142(2):393–406. [PMC free article] [PubMed]
  • Xu Z, Mayer A, Muller E, Wickner W. A heterodimer of thioredoxin and I(B)2 cooperates with Sec18p (NSF) to promote yeast vacuole inheritance. J Cell Biol. 1997 Jan 27;136(2):299–306. [PMC free article] [PubMed]
  • Yamakawa H, Seog DH, Yoda K, Yamasaki M, Wakabayashi T. Uso1 protein is a dimer with two globular heads and a long coiled-coil tail. J Struct Biol. 1996 May-Jun;116(3):356–365. [PubMed]

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