β and γ subunits of a yeast guanine nucleotide-binding protein are not essential for membrane association of the α subunit but are required for receptor coupling

Conditions were devised to demonstrate GTP-regulated coupling between the yeast S7E2-encoded receptor and its cognate guanine nucleotide-binding protein (G protein). Treatment of partially purified membranes with guanosine 5′-[γ-thio]triphosphate (GTP[γ-S]) converted the receptor from a high-affinity state (K_d = 17 nM) to a much lower affinity state (K_d ≈ 150 nM), as judged by three independent criteria: rate of ligand (α-factor) dissociation, equilibrium binding, and antagonist competition. Expression of STE2 from the GAL1 promoter in MATa/MATα diploids, which do not express GPA1 (encoding G protein α subunit, G_a ), STE4 (encoding G protein βsubunit, G_β), and STE18 (encoding G protein γ subunit, G_γ) but do express another G protein α subunit (product of GPA2), yielded a single class of low-affinity receptors that were GTP[γ-S]-insensitive, indicating that STE2 gene product cannot couple productively with other G proteins, even in the absence of competition by its conate 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_β and G_γ subunits are essential for foemation of a productive complex between a G_α subunit and its corresponding receptor. Wild-type STE4 and STE18 gene protucts were not essential for membrane localization of the GPA1 gene product, as indicated by cell fractionation and immunological analyses, suggesting that G_β and G_γ subunits interact with the receptor or make the G_α subunit competent to associate correctly with the receptor, or both. (.