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Mol. Cell. Biol., Sep 1996, 5194-5209, Vol 16, No. 9
HG Dohlman, J Song, D Ma, WE Courchesne and J Thorner
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.
Copyright © 1996, American Society for Microbiology
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)
Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA.
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