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 high-molecular-weight GTP-binding proteins that are involved in diverse cellular processes. The Vps1 protein (Vps1p) was shown to perform an essential function in the yeast secretory pathway. Here, we report the isolation and characterization of a mutant allele of the VPS1 gene, causing a dominant-negative vacuolar protein sorting (vps) defect, as demonstrated by the mislocalization of the vacuolar hydrolase carboxypeptidase Y (CPY). DNA sequence analysis of the mutant vps1 allele (vps1d-293) revealed a single point mutation, resulting in an amino acid exchange at position 293 from Ala to Asp. The mutation is located downstream of the tripartite GTP-binding motif found in the amino-terminal half of the protein. The observation that expression of wild-type Vps1p partially suppressed the dominant-negative CPY sorting phenotype indicates competition of a non-functional mutant Vps1 protein and a functional wild-type VPS1p for a Vps1p-binding site of an as yet unknown vacuolar protein sorting factor.
Finken-Eigen
M
M
Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, Germany.
Müller
S
S
Köhrer
K
K
D000409:Alanine / Q000737:chemistry; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D005800:Genes, Fungal; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D033921:Vesicular Transport Proteins; D001216:Asparagine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; C064553:VPS1 protein, S cerevisiae; D000409:Alanine