Beta and gamma subunits of a yeast guanine nucleotide-binding protein are not essential for membrane association of the alpha subunit but are required for receptor coupling.
Conditions were devised to demonstrate GTP-regulated coupling between the yeast STE2-encoded receptor and its cognate guanine nucleotide-binding protein (G protein). Treatment of partially purified membranes with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) converted the receptor from a high-affinity state (Kd = 17 nM) to a much lower affinity state (Kd approximately 150 nM), as judged by three independent criteria: rate of ligand (alpha-factor) dissociation, equilibrium binding, and antagonist competition. Expression of STE2 from the GAL1 promoter in MATa/MAT alpha diploids, which do not express GPA1 (encoding G protein alpha subunit, G alpha), STE4 (encoding G protein beta subunit, G beta), and STE18 (encoding G protein gamma subunit, G gamma) but do express another G protein alpha subunit (product of GPA2), yielded a single class of low-affinity receptors that were GTP[gamma-S]-insensitive, indicating that STE2 gene product cannot couple productively with other G proteins, even in the absence of competition by its cognate G protein. By using gpa1, STE4, and ste18 mutations, it was found that all three G protein subunits were required for functional coupling, as judged by the absence of high-affinity receptors when any of the three gene products was altered. This finding demonstrates that G beta and G gamma subunits are essential for formation of a productive complex between a G alpha subunit and its corresponding receptor. Wild-type STE4 and STE18 gene products were not essential for membrane localization of the GPA1 gene product, as indicated by cell fractionation and immunological analyses, suggesting that G beta and G gamma subunits interact with the receptor or make the G alpha subunit competent to associate correctly with the receptor, or both.
Blumer
K J
KJ
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Thorner
J
J
D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology / Q000648:ultrastructure*; D005800:Genes, Fungal; D046911:Macromolecular Substances; D000072235:Mating Factor; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000502:physiology*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014157:Transcription Factors
D046911:Macromolecular Substances; D010455:Peptides; D011956:Receptors, Cell Surface; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor; D019204:GTP-Binding Proteins