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PMID: 8106544 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

The Journal of cell biology ·Vol. 124 ·No. 4 ·1994-02-00 ·Pages 425-34

Oka T, Nakano A

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.

Related Genes
SAR1
MeSH Terms
Biological Transport Endoplasmic Reticulum/metabolism GTP-Binding Proteins/metabolism Golgi Apparatus/metabolism Guanosine 5'-O-(3-Thiotriphosphate)/metabolism Guanosine Triphosphate/metabolism Hydrolysis Kinetics Monomeric GTP-Binding Proteins Saccharomyces cerevisiae/metabolism Saccharomyces cerevisiae Proteins Temperature Vesicular Transport Proteins
Chemicals
Saccharomyces cerevisiae Proteins Vesicular Transport Proteins Guanosine 5'-O-(3-Thiotriphosphate) Guanosine Triphosphate GTP-Binding Proteins Monomeric GTP-Binding Proteins SAR1 protein, S cerevisiae
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Oka T
Department of Plant Sciences, Graduate School of Science, University of Tokyo, Japan.
Nakano A
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32 references, click to expand
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.124.4.425
Published
1994-02-00
Pages
425-34
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2119918
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.124.4.425 PMC Full Text View journal details
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