Biochemical investigation of protein transport in numerous experimental
systems has identified members of the SNARE, Sec1p, and Rab families
involved in the docking and fusion of transport vesicles to target
membranes. A genetic screen to isolate genes required for vacuolar
morphogenesis in Saccharomyces cerevisiae identified VAM7 ,
whose gene product contains a predicted coiled-coil domain homologous to
the t-SNARE SNAP-25 (Wada and Anraku, 1997. J. Biol. Chem.
267:18671-5). Analysis of a temperature-sensitive allele
vam7 tsf suggests that the VAM7 gene product
functions in endosome-to-vacuole protein transport. The
vam7 tsf mutant incubated at nonpermissive temperature
displayed a rapid defect in the trafficking of multiple vacuolar
proteins. Furthermore, Vam7p was found to interact directly with the
vacuolar syntaxin homolog Vam3p, suggesting that it functions in an
analogous manner to SNAP-25. Like Vam3p, a fraction of Vam7p localized
to vacuolar membranes. The trafficking defects of the
vam7 tsf mutant were suppressed by overexpression of
Vam3p, while vam7 tsf vam3 tsf double
mutants exhibited synthetic protein trafficking defects. Additionally,
Vam7p contains a putative PX domain. Site-directed mutations within this
domain resulted in temperature-conditional protein sorting defects and
synthetic phenotypes when combined with the vam3 tsf
mutant, indicating 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 fusion of
multiple transport intermediates destined for the vacuole.
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