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PMID: 9201718 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast.

Molecular biology of the cell ·Vol. 8 ·No. 6 ·1997-06-00 ·Pages 1089-104

Burd CG, Peterson M, Cowles CR, Emr SD

Abstract

The vacuolar protein-sorting (VPS) pathway of Saccharomyces cerevisiae mediates localization of proteins from the trans-Golgi to the vacuole via a prevacuolar endosome compartment. Mutations in class D vacuolar protein-sorting (vps) genes affect vesicle-mediated Golgi-to-endosome transport and result in secretion of vacuolar proteins. Temperature-sensitive-for-function (tsf) and dominant negative mutations in PEP12, encoding a putative SNARE vesicle receptor on the endosome, and tsf mutations in VAC1, a gene implicated in vacuole inheritance and vacuolar protein sorting, were constructed and used to demonstrate that Pep12p and Vac1p are components of the VPS pathway. The sequence of Vac1p contains two putative zinc-binding RING motifs, a zinc finger motif, and a coiled-coil motif. Site-directed mutations in the carboxyl-terminal RING motif strongly affected vacuolar protein sorting. Vac1p was found to be tightly associated with membranes as a monomer and in a large SDS-resistant complex. By using Pep12p affinity chromatography, we found that Vac1p, Vps45p (SEC1 family member), and Sec18p (yeast N-ethyl maleimide-sensitive factor, NSF) bind Pep12p. Consistent with a functional role for this complex in vacuolar protein sorting, double pep12tsfvac1tsf and pep12tsf vps45tsf mutants exhibited synthetic Vps- phenotypes, the tsf phenotype of the vac1tsf mutant was rescued by overexpression of VPS45 or PEP12, overexpression of a dominant pep12 allele in a sec18-1 strain resulted in a severe synthetic growth defect that was rescued by deletion of PEP12 or VAC1, and subcellular fractionation of vac1 delta cells revealed a striking change in the fractionation of Pep12p and Vps21p, a rab family GTPase required for vacuolar protein sorting. The functions of Pep12p, Vps45p, and Vps21p indicate that key aspects of Golgi-to-endosome trafficking are similar to other vesicle-mediated transport steps, although the role of Vac1p suggests that there are also novel components of the VPS pathway.

MeSH Terms
Adaptor Proteins, Signal Transducing Adenosine Triphosphatases Biological Transport Cell Compartmentation Cytoskeletal Proteins Endosomes/metabolism Fungal Proteins/genetics,metabolism,physiology GTP-Binding Proteins/metabolism Golgi Apparatus/metabolism Macromolecular Substances Membrane Fusion Membrane Proteins/metabolism Protein Binding Qa-SNARE Proteins Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins Structure-Activity Relationship Vacuoles/metabolism Vesicular Transport Proteins Zinc/metabolism rab GTP-Binding Proteins
Chemicals
Adaptor Proteins, Signal Transducing Cytoskeletal Proteins Fungal Proteins Macromolecular Substances Membrane Proteins PEP12 protein, S cerevisiae PEP7 protein, S cerevisiae Qa-SNARE Proteins Saccharomyces cerevisiae Proteins VPS45 protein, S cerevisiae Vesicular Transport Proteins Adenosine Triphosphatases GTP-Binding Proteins SEC18 protein, S cerevisiae rab GTP-Binding Proteins Zinc
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Burd C G
Division of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla 92093-0668, USA.
Peterson M
Cowles C R
Emr S D
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.8.6.1089
Published
1997-06-00
Pages
1089-104
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC305716
Subset
IM
Grants
NCI NIH HHS · CA-58689 · United States
NIGMS NIH HHS · GM-32703 · United States
NHLBI NIH HHS · HL-07491 · United States
External Links
PubMed source DOI: 10.1091/mbc.8.6.1089 PMC Full Text View journal details
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