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Proc Natl Acad Sci U S A. 1990 Jun;87(11):4363-7.

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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  • 1Department of Molecular and Cell Biology, University of California, Berkeley 94720.

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 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.

PMID:
2161538
PMCID:
PMC54110
[PubMed - indexed for MEDLINE]
Free PMC Article
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