Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Feb 2;124(4):425–434. doi: 10.1083/jcb.124.4.425

Inhibition of GTP hydrolysis by Sar1p causes accumulation of vesicles that are a functional intermediate of the ER-to-Golgi transport in yeast

PMCID: PMC2119918  PMID: 8106544

Abstract

The SAR1 gene product (Sar1p), a 21-kD GTPase, is a key component of the ER-to-Golgi transport in the budding yeast. We previously reported that the in vitro reconstitution of protein transport from the ER to the Golgi was dependent on Sar1p and Sec12p (Oka, T., S. Nishikawa, and A. Nakano. 1991. J. Cell Biol. 114:671-679). Sec12p is an integral membrane protein in the ER and is essential for the Sar1 function. In this paper, we show that Sar1p can remedy the temperature-sensitive defect of the sec12 mutant membranes, which is in the formation of ER- to-Golgi transport vesicles. The addition of Sar1p promotes vesicle formation from the ER irrespective of the GTP- or GTP gamma S-bound form, indicating that the active form of Sar1p but not the hydrolysis of GTP is required for this process. The inhibition of GTP hydrolysis blocks transport of vesicles to the Golgi and thus causes their accumulation. The accumulating vesicles, which carry Sar1p on them, can be separated from other membranes, and, after an appropriate wash that removes Sar1p, are capable of delivering the content to the Golgi when added back to fresh membranes. Thus we have established a new method for isolation of functional intermediate vesicles in the ER-to-Golgi transport. The sec23 mutant is defective in activation of Sar1 GTPase (Yoshihisa, T., C. Barlowe, and R. Schekman. 1993. Science (Wash. DC). 259:1466-1468). The membranes and cytosol from the sec23 mutant show only a partial defect in vesicle formation and this defect is also suppressed by the increase of Sar1p. Again GTP hydrolysis is not needed for the suppression of the defect in vesicle formation. Based on these results, we propose a model in which Sar1p in the GTP-bound form is required for the formation of transport vesicles from the ER and the GTP hydrolysis by Sar1p is essential for entering the next step of vesicular transport to the Golgi apparatus.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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. doi: 10.1016/0092-8674(88)90196-1. [DOI] [PubMed] [Google Scholar]
  2. Balch W. E. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 1990 Dec;15(12):473–477. doi: 10.1016/0968-0004(90)90301-q. [DOI] [PubMed] [Google Scholar]
  3. Beckers C. J., Balch W. E. Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J Cell Biol. 1989 Apr;108(4):1245–1256. doi: 10.1083/jcb.108.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Franzusoff A., Lauzé E., Howell K. E. Immuno-isolation of Sec7p-coated transport vesicles from the yeast secretory pathway. Nature. 1992 Jan 9;355(6356):173–175. doi: 10.1038/355173a0. [DOI] [PubMed] [Google Scholar]
  5. Goud B., Salminen A., Walworth N. C., Novick P. J. 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. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  6. Groesch M. E., Ruohola H., Bacon R., Rossi G., Ferro-Novick S. Isolation of a functional vesicular intermediate that mediates ER to Golgi transport in yeast. J Cell Biol. 1990 Jul;111(1):45–53. doi: 10.1083/jcb.111.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hicke L., Schekman R. Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro. EMBO J. 1989 Jun;8(6):1677–1684. doi: 10.1002/j.1460-2075.1989.tb03559.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hosobuchi M., Kreis T., Schekman R. SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer. Nature. 1992 Dec 10;360(6404):603–605. doi: 10.1038/360603a0. [DOI] [PubMed] [Google Scholar]
  9. Kaiser C. A., 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. doi: 10.1016/0092-8674(90)90483-u. [DOI] [PubMed] [Google Scholar]
  10. Lian J. P., 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. doi: 10.1016/0092-8674(93)90253-m. [DOI] [PubMed] [Google Scholar]
  11. Lodish H. F., Kong N., Hirani S., Rasmussen J. A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex. J Cell Biol. 1987 Feb;104(2):221–230. doi: 10.1083/jcb.104.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Melançon P., Glick B. S., Malhotra V., Weidman P. J., Serafini T., Gleason M. L., Orci L., Rothman J. E. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. doi: 10.1016/0092-8674(87)90591-5. [DOI] [PubMed] [Google Scholar]
  13. Nakano A., Brada D., Schekman R. A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J Cell Biol. 1988 Sep;107(3):851–863. doi: 10.1083/jcb.107.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nakańo A., Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677–2691. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Newman A. P., Graf J., Mancini P., Rossi G., Lian J. P., Ferro-Novick S. SEC22 and SLY2 are identical. Mol Cell Biol. 1992 Aug;12(8):3663–3664. doi: 10.1128/mcb.12.8.3663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nishikawa S., Nakano A. Identification of a gene required for membrane protein retention in the early secretory pathway. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8179–8183. doi: 10.1073/pnas.90.17.8179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nishikawa S., Nakano A. The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway. Biochim Biophys Acta. 1991 Jul 10;1093(2-3):135–143. doi: 10.1016/0167-4889(91)90114-d. [DOI] [PubMed] [Google Scholar]
  18. Oka T., Nishikawa S., Nakano A. Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport. J Cell Biol. 1991 Aug;114(4):671–679. doi: 10.1083/jcb.114.4.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ossig R., Dascher C., Trepte H. H., Schmitt H. D., Gallwitz D. The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport. Mol Cell Biol. 1991 Jun;11(6):2980–2993. doi: 10.1128/mcb.11.6.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  21. Paulik M., Nowack D. D., Morré D. J. Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver. J Biol Chem. 1988 Nov 25;263(33):17738–17748. [PubMed] [Google Scholar]
  22. Pfeffer S. R. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. doi: 10.1016/0962-8924(92)90161-f. [DOI] [PubMed] [Google Scholar]
  23. Pryer N. K., Salama N. R., Schekman R., Kaiser C. A. Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro. J Cell Biol. 1993 Feb;120(4):865–875. doi: 10.1083/jcb.120.4.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rexach M. F., Schekman R. W. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol. 1991 Jul;114(2):219–229. doi: 10.1083/jcb.114.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ruohola H., Kabcenell A. K., Ferro-Novick S. Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant. J Cell Biol. 1988 Oct;107(4):1465–1476. doi: 10.1083/jcb.107.4.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  27. Schmitt H. D., Puzicha M., Gallwitz D. Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium. Cell. 1988 May 20;53(4):635–647. doi: 10.1016/0092-8674(88)90579-x. [DOI] [PubMed] [Google Scholar]
  28. 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. doi: 10.1126/science.1904626. [DOI] [PubMed] [Google Scholar]
  29. 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. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  30. Yoshihisa T., Barlowe C., Schekman R. Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum. Science. 1993 Mar 5;259(5100):1466–1468. doi: 10.1126/science.8451644. [DOI] [PubMed] [Google Scholar]
  31. d'Enfert C., Barlowe C., Nishikawa S., Nakano A., Schekman R. Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum. Mol Cell Biol. 1991 Nov;11(11):5727–5734. doi: 10.1128/mcb.11.11.5727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. d'Enfert C., Wuestehube L. J., Lila T., Schekman R. Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER. J Cell Biol. 1991 Aug;114(4):663–670. doi: 10.1083/jcb.114.4.663. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES