Mutations in the SHR5 gene of Saccharomyces cerevisiae suppress Ras function and block membrane attachment and palmitoylation of Ras proteins.
We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins.
Jung
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V
Cold Spring Harbor Laboratory, New York 11724.
Chen
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L
Hofmann
S L
SL
Wigler
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M
Powers
S
S
D000217:Acyltransferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D000939:Epitopes / Q000032:analysis; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005800:Genes, Fungal; D005838:Genotype; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic; D013696:Temperature; D014633:Valine; D018631:ras Proteins
D017931:DNA Primers; D000939:Epitopes; D005656:Fungal Proteins; D010169:Palmitic Acids; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019308:Palmitic Acid; D000217:Acyltransferases; C092131:SHR5 protein, S cerevisiae; D019204:GTP-Binding Proteins; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D014633:Valine