The Leu-132 of the Ste4(Gbeta) subunit is essential for proper coupling of the G protein with the Ste2 alpha factor receptor during the mating pheromone response in yeast.
In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits.
Ongay-Larios
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Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, P.O. Box 70-242, 04510, Mexico, Mexico.
Saviñón-Tejeda
A L
AL
Williamson
M J
MJ
Durán-Avelar
M d
Md
Coria
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D019943:Amino Acid Substitution / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D005656:Fungal Proteins / Q000235:genetics; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D000072235:Mating Factor; D008565:Membrane Proteins; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000494:pharmacology*; D010675:Pheromones / Q000494:pharmacology; D011485:Protein Binding; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000187:drug effects; D020798:Two-Hybrid System Techniques
C490024:FUS1 protein, S cerevisiae; D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D008565:Membrane Proteins; D010455:Peptides; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D014157:Transcription Factors; D000072235:Mating Factor; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D007930:Leucine