Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit)

Mol Cell Biol. 1996 Sep;16(9):5194-209. doi: 10.1128/MCB.16.9.5194.

Abstract

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cattle
  • Cell Division
  • Escherichia coli / genetics
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / physiology*
  • GTP-Binding Protein alpha Subunits*
  • GTP-Binding Protein alpha Subunits, Gq-G11
  • GTP-Binding Proteins / antagonists & inhibitors*
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism
  • GTPase-Activating Proteins*
  • Gene Expression Regulation, Fungal*
  • Heterotrimeric GTP-Binding Proteins*
  • Humans
  • Macromolecular Substances
  • Mating Factor
  • Molecular Sequence Data
  • Peptides / physiology*
  • Receptors, Mating Factor
  • Receptors, Peptide / physiology
  • Recombinant Fusion Proteins / metabolism
  • Reproduction / physiology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae Proteins*
  • Signal Transduction
  • Transcription Factors*

Substances

  • Fungal Proteins
  • GTP-Binding Protein alpha Subunits
  • GTPase-Activating Proteins
  • Macromolecular Substances
  • Peptides
  • Receptors, Mating Factor
  • Receptors, Peptide
  • Recombinant Fusion Proteins
  • SST2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Mating Factor
  • GTP-Binding Proteins
  • GPA1 protein, S cerevisiae
  • GTP-Binding Protein alpha Subunits, Gq-G11
  • Heterotrimeric GTP-Binding Proteins