CLONING AND CHARACTERIZATION OF A DOMINANT-NEGATIVE VPS1 ALLELE OF THE YEAST SACCHAROMYCES-CEREVISIAE

The gene product of the yeast VPS1 gene is a member of a family of hig h-molecular-weight GTP-binding proteins that are involved in diverse c ellular processes. The Vps1 protein (Vps1p) was shown to perform an es sential function in the yeast secretory pathway. Here, we report the i solation and characterization of a mutant allele of the VPS1 gene, cau sing a dominant-negative vacuolar protein sorting (vps) defect, as dem onstrated by the mislocalization of the vacuolar hydrolase carboxypept idase Y (CPY). DNA sequence analysis of the mutant vps1 allele (vps1(d )-293) revealed a single point mutation, resulting in an amino acid ex change at position 293 from Ala to Asp. The mutation is located downst ream of the tripartite GTP-binding motif found in the amino-terminal h alf of the protein. The observation that expression of wild-type Vps1p partially suppressed the dominant-negative CPY sorting phenotype indi cates competition of a non-functional mutant Vps1 protein and a functi onal wild-type VPS1p for a Vps1p-binding site of an as yet unknown vac uolar protein sorting factor.