Biochemical analysis of yeast G(alpha) mutants that enhance adaptation to pheromone.
The mating-specific heterotrimeric G(alpha) protein of Saccharomyces cerevisiae, Gpa1, negatively regulates activation of the pheromone response pathway both by sequestering G(beta)gamma and by triggering an adaptive response through an as yet unknown mechanism. Previous genetic studies identified mutant alleles of GPA1 that downregulate the pheromone response independently of the pheromone receptor (GPA1E364K), or through a receptor-dependent mechanism (GPA1N388D). To further our understanding of the mechanism of action of these mutant alleles, their corresponding proteins were purified and subjected to biochemical analysis. The receptor-dependent activity of Gpa1N388D was further analyzed using yeast strains expressing constitutively active receptor (Ste2) mutants, and C-terminal truncation mutant forms of Gpa1. A combination of G(alpha) affinity chromatography, GTP binding/hydrolysis studies, and genetic analysis allowed us to assign a distinct mechanism of action to each of these mutant proteins.
Cismowski
M J
MJ
Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Metodiev
M
M
Draper
E
E
Stone
D E
DE
D000222:Adaptation, Physiological / Q000502:physiology*; D000483:Alleles; D017607:Aluminum Compounds / Q000737:chemistry; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D002846:Chromatography, Affinity; D015536:Down-Regulation / Q000187:drug effects; D005459:Fluorides / Q000737:chemistry; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010675:Pheromones / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014157:Transcription Factors
D017607:Aluminum Compounds; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010675:Pheromones; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C084294:STE18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D014157:Transcription Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D006160:Guanosine Triphosphate; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride