An arf1Delta synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport in Saccharomyces cerevisiae.
ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
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Chen
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Graham
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000469:Alkaline Phosphatase / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D001692:Biological Transport; D002268:Carboxypeptidases / Q000378:metabolism*; D043402:Cathepsin A; D002966:Clathrin / Q000096:biosynthesis / Q000235:genetics*; D033922:Clathrin Heavy Chains; D016368:Frameshift Mutation; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005800:Genes, Fungal / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D005816:Genetic Complementation Test; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000201:enzymology*; D043324:beta-Fructofuranosidase
D002966:Clathrin; D029701:Saccharomyces cerevisiae Proteins; D033922:Clathrin Heavy Chains; D000469:Alkaline Phosphatase; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D002268:Carboxypeptidases; D043402:Cathepsin A; C019836:CPS1 protein, S cerevisiae; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors