Site-directed mutations altering the CAAX box of Ste18, the yeast pheromone-response pathway G gamma subunit.
The STE18 gene encodes the gamma subunit of the G protein which functions in the Saccharomyces cerevisiae pheromone-response pathway. The STE18 gene product undergoes a post-translational processing at the carboxyl terminus directed by the CCAAX box motif CCTLM110. A variety of site-directed mutations of this sequence have been constructed to test the role of this motif on Ste18 function. Mutations which change or eliminate the cysteine at position 107 abolish Ste18-dependent mating, and thus the cysteine (C107) is essential for Ste18 function. However, inactivation of the prenyltransferase by disruption of DPR1 has only a minor effect on Ste18-dependent mating. Mutation of cysteine 106 to serine significantly reduces but does not eliminate Ste18 function. Deletion of the C-terminal TLM sequence or modification of the ultimate methionine to lysine, arginine or leucine, all changes which do not affect the CAAX box cysteines, have only minor effects on Ste18-dependent mating. Intriguingly, these latter mutations dramatically compromise Ste18 function in cells which are deleted for Gpa1, the alpha subunit of the G protein. In addition, overexpression of these mutant versions of STE18 causes a dominant negative phenotype and inhibits the constitutive mating response generated by GPA1 deletion in cells which contain a functional STE18 gene. These results suggest that the C terminus of Ste18 and the Gpa1 protein have overlapping roles in some aspect of yeast G protein function such as membrane targeting.
Whiteway
M S
MS
Biotechnology Research Institute, National Research Council of Canada, Montreal.
Thomas
D Y
DY
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D051231:Farnesyltranstransferase; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology; D005800:Genes, Fungal; D051228:Geranyltranstransferase; D020962:Heterotrimeric GTP-Binding Proteins; D000072235:Mating Factor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000502:physiology*; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D014166:Transferases / Q000378:metabolism
D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D044388:GTP-Binding Protein gamma Subunits; D010455:Peptides; C084294:STE18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase; D051231:Farnesyltranstransferase; D019204:GTP-Binding Proteins; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins