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.

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Abstract
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 .apprxeq. 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.