Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae: roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network

• Published in: Genetics (Austin) Vol. 154; no. 1; pp. 83 - 97
• Main Authors: Bensen, E.S, Costaguta, G, Payne, G.S
• Format: Journal Article
• Language: English
• Published: United States Genetics Soc America 2000; Genetics Society of America
• Subjects: alpha -factor; anatomy and morphology; Base Sequence; binding proteins; carboxypeptidases; Carrier Proteins - physiology; CHC1 gene; Clathrin - metabolism; DNA Primers; dynamin; Endocytosis; gene interaction; genes; Genes, Synthetic; Genetics; Golgi Apparatus - metabolism; GTP-Binding Proteins; inositol polyphosphate 5-phosphatase; Inositol Polyphosphate 5-Phosphatases; inositol polyphosphate 5-phosphatse; INP52 gene; INP53 gene; Inp53 protein; Inp53p protein; LUV1 gene; Mutagenesis; mutants; mutation; phenotype; pheromones; phosphoprotein phosphatase; phosphoric monoester hydrolases; Phosphoric Monoester Hydrolases - physiology; protein transport; Proteins; PTC1 gene; RIC1 gene; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; synaptojanin; synthetic genes; Temperature; vacuoles; Vesicular Transport Proteins; VPS1 gene; Vps1p protein; Yeast
• ISSN: 0016-6731; 1943-2631; 1943-2631
• DOI: 10.1093/genetics/154.1.83

Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.