Dual lipid modification of the yeast ggamma subunit Ste18p determines membrane localization of Gbetagamma.
The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gbetagamma subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gbetagamma complex from cells cultured in the presence of [(3)H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Ggamma and Gbetagamma. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gbetagamma to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gbetagamma depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.
Hirschman
J E
JE
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122, USA.
Jenness
D D
DD
D002451:Cell Compartmentation; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism; D010169:Palmitic Acids; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D005231:Fatty Acids, Unsaturated; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008565:Membrane Proteins; D010169:Palmitic Acids; C084294:STE18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D019308:Palmitic Acid; C007702:farnesoic acid; D020962:Heterotrimeric GTP-Binding Proteins