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Molecular and Cellular Biology, September 1998, p. 5308-5319, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Vam7p, a SNAP-25-Like Molecule, and Vam3p, a Syntaxin Homolog, Function Together in Yeast Vacuolar Protein Trafficking

Trey K. Sato, Tamara Darsow, and Scott D. Emr*

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

Received 20 April 1998/Returned for modification 1 June 1998/Accepted 15 June 1998

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 (vam7tsf) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7tsf 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 vam7tsf 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 p40phox (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3tsf 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.

* Corresponding author. Mailing address: Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego School of Medicine, La Jolla, CA 92093-0668. Phone: 619-534-6462. Fax: 619-534-6414. E-mail: semr{at}ucsd.edu.

Molecular and Cellular Biology, September 1998, p. 5308-5319, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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