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

Kendall J. Blumer, Jeremy Thorner

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78 Scopus citations

Abstract

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 (Kd = 17 nM) to a much lower affinity state (Kd ≈ 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, Ga ), STE4 (encoding G protein βsubunit, Gβ), and STE18 (encoding G protein γ subunit, Gγ) 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 Gβ and Gγ subunits are essential for foemation of a productive complex between a Gα 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 Gβ and Gγ subunits interact with the receptor or make the Gα subunit competent to associate correctly with the receptor, or both. (.

Original languageEnglish
Pages (from-to)4363-4367
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number11
DOIs
StatePublished - 1990

Keywords

  • Guanosine 5′-[γ-thio]triphosphate
  • Peptide hormone
  • Saccharomyces cerevisiae
  • Signal transduction

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