SST2, A NEGATIVE REGULATOR OF PHEROMONE SIGNALING IN THE YEAST SACCHAROMYCES-CEREVISIAE - EXPRESSION, LOCALIZATION, AND GENETIC INTERACTIONAND PHYSICAL ASSOCIATION WITH GPA1 (THE G-PROTEIN ALPHA-SUBUNIT)

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST 2 gene product fail to resume growth after exposure to pheromone. Conv ersely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detec tably dampened signaling in a short-term assay of pheromone response ( phosphorylation of Ste4, G beta subunit). When the GPA1 gene product ( G alpha subunit) is absent, the pheromone response pathway is constitu tively active and, consequently, growth ceases, Despite sustained indu ction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1 Delt a mutants remain growth arrested, indicating that the action of Sst2 r equires the presence of Gpa1. The N-terminal domain (residues 3 to 307 ) of Sst2 (698 residues) has sequence similarity to the catalytic regi ons of bovine GTPase-activating protein and human neurofibromatosis tu mor suppressor protein; segments in the C-terminal domain of Sst2 (bet ween 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 acti vity of Gpa1, the majority of Sst2 was membrane associated and colocal ized with Gpa1 at the plasma membrane, as judged by sucrose density gr adient fractionation. Moreover, from cell extracts, Sst2 could be isol ated 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, S ST2(+) cells expressing a GTPase-defective GPA1 mutant displayed an in creased sensitivity to pheromone, whereas sst2 cells did not. These re sults demonstrate that Sst2 and Gpa1 interact physically and suggest t hat Sst2 is a direct negative regulator of Gpa1.