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PMID: 9710615 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together in yeast vacuolar protein trafficking.

Molecular and cellular biology ·Vol. 18 ·No. 9 ·1998-09-00 ·Pages 5308-19

Sato TK, Darsow T, Emr SD

Abstract

A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671-18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046-3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf)) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7(tsf) mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination of vam7(tsf) cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40(phox) (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3(tsf) mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.

MeSH Terms
Amino Acid Sequence Carrier Proteins/metabolism Cell Fractionation Fungal Proteins/metabolism Genotype Intracellular Membranes/metabolism,ultrastructure Membrane Proteins/chemistry,metabolism Microscopy, Electron Models, Biological Molecular Sequence Data N-Ethylmaleimide-Sensitive Proteins Nerve Tissue Proteins/chemistry,genetics,metabolism Point Mutation Qa-SNARE Proteins Qc-SNARE Proteins Saccharomyces cerevisiae/genetics,physiology,ultrastructure Saccharomyces cerevisiae Proteins Sequence Alignment Sequence Homology, Amino Acid Synaptosomal-Associated Protein 25 Temperature Vacuoles/physiology,ultrastructure Vesicular Transport Proteins
Chemicals
Carrier Proteins Fungal Proteins Membrane Proteins Nerve Tissue Proteins Qa-SNARE Proteins Qc-SNARE Proteins Saccharomyces cerevisiae Proteins Synaptosomal-Associated Protein 25 VAM3 protein, S cerevisiae VAM7 protein, S cerevisiae Vesicular Transport Proteins N-Ethylmaleimide-Sensitive Proteins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Sato T K
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California at San Diego School of Medicine, La Jolla, California 92093-0668, USA.
Darsow T
Emr S D
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.18.9.5308
Published
1998-09-00
Pages
5308-19
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC109116
Subset
IM
Grants
NCI NIH HHS · CA58689 · United States
External Links
PubMed source DOI: 10.1128/MCB.18.9.5308 PMC Full Text View journal details
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