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PMID: 9245783 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole.

The Journal of cell biology ·Vol. 138 ·No. 3 ·1997-08-11 ·Pages 517-29

Darsow T, Rieder SE, Emr SD

Abstract

Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283-amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3(tsf)). Upon shifting the vam3(tsf) mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3(tsf) cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3(tsf) cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Delta mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Delta mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

MeSH Terms
Aminopeptidases/metabolism Autophagy Biological Transport Carboxypeptidases/metabolism Carrier Proteins Cathepsin A Cell Compartmentation Cytoplasm/metabolism Fungal Proteins/genetics,metabolism Golgi Apparatus/metabolism Hydrolases/metabolism Membrane Fusion Membrane Proteins/chemistry,genetics,metabolism Mutation Phagosomes/metabolism,ultrastructure Qa-SNARE Proteins Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins Temperature Vacuoles/metabolism,ultrastructure Vesicular Transport Proteins
Chemicals
Carrier Proteins Fungal Proteins Membrane Proteins PEP12 protein, S cerevisiae Qa-SNARE Proteins Saccharomyces cerevisiae Proteins VPS33 protein, S cerevisiae Vesicular Transport Proteins Hydrolases Carboxypeptidases Aminopeptidases APE1 protein, S cerevisiae Cathepsin A
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Darsow T
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668, USA.
Rieder S E
Emr S D
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.138.3.517
Published
1997-08-11
Pages
517-29
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2141632
Subset
IM
Grants
NCI NIH HHS · CA58689 · United States
NIGMS NIH HHS · GM32703 · United States
External Links
PubMed source DOI: 10.1083/jcb.138.3.517 PMC Full Text View journal details
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