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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
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 (vam3tsf). Upon shifting the
vam3tsf 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, vam3tsf 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,
vam3tsf 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 vam3
mutant phenotypes and, likewise, overexpression of
Vam3p suppressed the pep12 mutant phenotypes.
This result indicated that SNAREs alone do not define
the specificity of vesicle docking reactions.
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