Genome-Scale Analysis Reveals Sst2 as the Principal Regulator of Mating Pheromone Signaling in the Yeast Saccharomyces cerevisiae

Abstract

A common property of G protein-coupled receptors is that they become less responsive with prolonged stimulation. Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity and do so by stabilizing the transition state conformation of the G protein ␣ subunit. In the yeast Saccharomyces cerevisiae there are four RGS-homologous proteins (Sst2, Rgs2, Rax1, and Mdm1) and two G␣ proteins (Gpa1 and Gpa2). We show that Sst2 is the only RGS protein that binds selectively to the transition state conformation of Gpa1. The other RGS proteins also bind Gpa1 and modulate pheromone signaling, but to a lesser extent and in a manner clearly distinct from Sst2. To identify other candidate pathway regulators, we compared pheromone responses in 4,349 gene deletion mutants representing nearly all nonessential genes in yeast. A number of mutants produced an increase (sst2, bar1, asc1, and ygl024w) or decrease (cla4) in pheromone sensitivity or resulted in pheromone-independent signaling (sst2, pbs2, gas1, and ygl024w). These findings suggest that Sst2 is the principal regulator of Gpa1-mediated signaling in vivo but that other proteins also contribute in distinct ways to pathway regulation.G protein-coupled receptors respond to a vast array of chemical and sensory signals, including hormones, neurotransmitters, odors, and light. Approximately one-third of all drugs act by binding directly to receptors of this class (64). Upon agonist stimulation of the receptor, a cognate G protein ␣ subunit will exchange GDP for GTP and undergo dissociation from the G protein ␤␥ subunit dimer. The dissociated subunits bind to effector enzymes, which in turn activate protein kinases, trigger new gene transcription, and ultimately produce programmed changes in cell homeostasis or differentiation (90). Regulators of G protein signaling (RGS proteins) function as GTPase-accelerating proteins (GAPs) and, in this manner, promote rapid inactivation or desensitization of the signal (89).Whereas mammalian genome analysis has revealed at least 16 G␣-and ϳ40 RGS-encoding genes (89, 106), a similar analysis in the yeast Saccharomyces cerevisiae reveals only two G␣ subunits but at least four RGS protein homologues. Gpa1 mediates cellular responses to mating pheromones. These pheromones, called a-factor and ␣-factor, are produced by haploid a and ␣ cells and bind to G protein-coupled receptors on cells of the opposite mating type. Upon activation of pheromone receptors, Gpa1 binds to GTP and dissociates from the G␤␥ dimer Ste4/Ste18, and the dissociated subunits activate a multitude of downstream effectors leading to cell fusion (mating) to form an a/␣ diploid (36, 50). Prominent among the known effectors are components of a MAP (mitogen-activated protein) kinase cascade comprised of Ste20, Ste11, Ste7, and Fus3. A parallel signaling pathway responds to glucose stimulation, leading to activation of a distinct receptor (Gpr1) (66,73,76,99,124), a distinct G protein ␣ subunit (Gpa2), and an atypical G protein ␤␥ complex ...

Global Analysis of Serine-Threonine Protein Kinase Genes in Neurospora crassa

“…Three mutants formed normal perithecia but ejected no (⌬nim-1), few (⌬mst-1), or white ascospores (⌬vel; 20% of ascospores were white.). One of the S. cerevisiae vel homologs, cla4, is required for normal pheromone sensitivity (10,41). This suggests that Cla4p acts at an earlier point in sexual development in yeast than VEL in Neurospora.…”

The RACK1 Ortholog Asc1 Functions as a G-protein β Subunit Coupled to Glucose Responsiveness in Yeast

“…matin structure (66,67). We have also shown previously that Asc1 interacts weakly with the G␣ in the pheromone response pathway, Gpa1, and deletion of ASC1 results in cells that are slightly more sensitive to pheromone stimulation (39). Such diversity of function has no precedent among the more "typical" G␤ subunits.…”

“…Yeast shuttle plasmids pRS315-ADH (CEN, amp R , LEU2, ADH1 promoter/terminator) and pRS316-ADH (CEN, amp R , URA3, ADH1 promoter/terminator) (38) were modified by PCR amplification of the gene of interest and subcloned such that a FLAG (GATTA-CAAGGATGACGACGATAAG) or Myc (GAACAAAAATT-GATTTCTGAAGAAGATTTG) epitope could be added to the 5Ј coding sequence of any gene with a SacI site engineered inframe with the open reading frame. Previously described yeast shuttle plasmids used in this study are pAD4M-GST (38) and pAD4M-GPA2-GST (39). The FRE-lacZ transcription reporter plasmid was generously provided by Gerald Fink (40).…”

Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling

“…Asc1p's involvement in cellular signal transduction prompted us to investigate Asc1p-sensitive protein phosphorylation beyond its own phosphorylation. Previous studies reported that the absence of Asc1p leads to increased phosphorylation of the MAPKs Kss1p and Slt2p, which control filamentous growth, mating, and cell wall integrity (2,29). In the present study, we additionally analyzed the phosphorylation of the MAPK of the high-osmolarity response pathway, Hog1p, by using an antibody specific for this protein phosphorylated at residues T174 and/or Y176 by its upstream mitogen-activated protein kinase kinase (MAP2K), Pbs2p.…”