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)

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.

[1]  M. Lohse,et al.  Molecular mechanisms of membrane receptor desensitization. , 1993, Biochimica et biophysica acta.

[2]  B. Haarer,et al.  Immunofluorescence methods for yeast. , 1991, Methods in enzymology.

[3]  S. Arkinstall,et al.  Yeast alpha-mating factor receptor-linked G-protein signal transduction suppresses Ras-dependent activity. , 1991, FEBS letters.

[4]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. A. Moore,et al.  Yeast cells recover from mating pheromone alpha factor-induced division arrest by desensitization in the absence of alpha factor destruction. , 1984, The Journal of biological chemistry.

[6]  F. Tamanoi,et al.  The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae , 1990, Cell.

[7]  R. W. Davis,et al.  Replacement of chromosome segments with altered DNA sequences constructed in vitro. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[8]  David Y. Thomas,et al.  The STE4 and STE18 genes of yeast encode potential β and γ subunits of the mating factor receptor-coupled G protein , 1989, Cell.

[9]  J. Kurjan.  The pheromone response pathway in Saccharomyces cerevisiae. , 1993, Annual review of genetics.

[10]  J. Armstrong,et al.  Characterization of the Bar proteinase, an extracellular enzyme from the yeast Saccharomyces cerevisiae. , 1991, Advances in experimental medicine and biology.

[11]  K. Clark,et al.  Genetic identification of residues involved in association of alpha and beta G-protein subunits , 1994, Molecular and cellular biology.

[12]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[13]  J. Thorner,et al.  Yeast α factor is processed from a larger precursor polypeptide: The essential role of a membrane-bound dipeptidyl aminopeptidase , 1983, Cell.

[14]  J. Thorner,et al.  Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae , 1983, Journal of bacteriology.

[15]  L. Lim,et al.  Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24 , 1995, Molecular and cellular biology.

[16]  M. Farquhar,et al.  GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Arai,et al.  Regulation of the yeast pheromone response pathway by G protein subunits. , 1990, The EMBO journal.

[18]  J. Broach,et al.  Ras membrane targeting is essential for glucose signaling but not for viability in yeast. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Whiteway,et al.  Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae. , 1990, Molecular and cellular biology.

[20]  G. Milligan,et al.  Agonist regulation of cellular G protein levels and distribution: mechanisms and functional implications. , 1993, Trends in pharmacological sciences.

[21]  J. Thorner,et al.  12 Pheromone Response and Signal Transduction during the Mating Process of Saccharomyces cerevisiae , 1992 .

[22]  W. J. Chen,et al.  The role of protein structure in the mitochondrial import pathway. Analysis of the soluble F1-ATPase beta-subunit precursor. , 1987, The Journal of biological chemistry.

[23]  R. Cerione,et al.  Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain. , 1993, The Journal of biological chemistry.

[24]  R. K. Chan,et al.  Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones , 1982, Molecular and cellular biology.

[25]  H. Dohlman,et al.  Regulation of Membrane and Subunit Interactions by N-Myristoylation of a G Protein α Subunit in Yeast* , 1996, The Journal of Biological Chemistry.

[26]  A. Gilman,et al.  Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha. , 1989, The Journal of biological chemistry.

[27]  R. Cerione,et al.  Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast , 1994, The Journal of cell biology.

[28]  M. Goebl,et al.  N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase. , 1991, Genes & development.

[29]  H. Dohlman,et al.  Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[30]  B. Ferguson,et al.  Signal transduction and growth control in yeast. , 1995, Current opinion in genetics & development.

[31]  R. Schekman,et al.  Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. , 1992, Molecular biology of the cell.

[32]  S. Pennington.  GTP-binding proteins 1: heterotrimeric G proteins. , 1995, Protein profile.

[33]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Botstein,et al.  Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. , 1993, Molecular biology of the cell.

[35]  M. Tyers,et al.  A new family of regulators of G-protein-coupled receptors? , 1996, Current Biology.

[36]  J. Kurjan,et al.  Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone. , 1987, Molecular and cellular biology.

[37]  J. Thorner,et al.  Intracellular targeting and structural conservation of a prohormone-processing endoprotease. , 1989, Science.

[38]  S. Reed,et al.  Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling , 1988, Molecular and cellular biology.

[39]  V. Arshavsky,et al.  Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP , 1992, Nature.

[40]  L. Hartwell,et al.  The C-terminus of the S. cerevisiae α-pheromone receptor mediates an adaptive response to pheromone , 1988, Cell.

[41]  L. Bardwell,et al.  Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae. , 1994, Developmental biology.

[42]  J. Kurjan,et al.  The yeast SCG1 gene: A Gα-like protein implicated in the a- and α-factor response pathway , 1987, Cell.

[43]  S. Reed,et al.  G protein mutations that alter the pheromone response in Saccharomyces cerevisiae , 1990, Molecular and cellular biology.

[44]  H. Bourne,et al.  Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin. , 1984, The Journal of biological chemistry.

[45]  G. Saari,et al.  The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[46]  E. Ross,et al.  Phospholipase C-β1 is a GTPase-activating protein for Gq/11, its physiologic regulator , 1992, Cell.

[47]  H. Heng,et al.  Differential expression of a basic helix-loop-helix phosphoprotein gene, G0S8, in acute leukemia and localization to human chromosome 1q31. , 1995, Leukemia.

[48]  S. Reed,et al.  Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway , 1990, Molecular and cellular biology.

[49]  G. Sprague,,et al.  Assay of yeast mating reaction. , 1991, Methods in enzymology.

[50]  J. Thorner,et al.  Recovery of S. cerevisiae a cells from G1 arrest by α factor pheromone requires endopeptidase action , 1979, Cell.

[51]  H. Horvitz,et al.  EGL-10 Regulates G Protein Signaling in the C. elegans Nervous System and Shares a Conserved Domain with Many Mammalian Proteins , 1996, Cell.

[52]  N. Walworth,et al.  A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast , 1988, Cell.

[53]  J. Thorner,et al.  Model systems for the study of seven-transmembrane-segment receptors. , 1991, Annual review of biochemistry.

[54]  J. Thorner,et al.  A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae , 1989, Cell.

[55]  S. Sprang,et al.  Mechanism of GTP hydrolysis by G-protein alpha subunits. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[56]  F. McCormick,et al.  The ras Superfamily of GTPases , 1993 .

[57]  J. Thorner,et al.  Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[58]  Margaret Robertson,et al.  The neurofibromatosis type 1 gene encodes a protein related to GAP , 1990, Cell.

[59]  P. Novick,et al.  Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle , 1991, The Journal of cell biology.

[60]  J. Thorner,et al.  G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover , 1994, Molecular and cellular biology.

[61]  R. K. Chan,et al.  Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones , 1982, Molecular and cellular biology.

[62]  G. Fink,et al.  Laboratory course manual for methods in yeast genetics , 1986 .

[63]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[64]  J. Hirsch,et al.  Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway. , 1991, Genes & development.

[65]  G. Sprague,,et al.  Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast , 1993, The Journal of cell biology.

[66]  J. Thorner,et al.  Identification of tubulin from the yeast Saccharomyces cerevisiae. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[67]  S. Reed,et al.  Pheromone-induced phosphorylation of a G protein β subunit in S. cerevisiae is associated with an adaptive response to mating pheromone , 1991, Cell.

[68]  J. Thorner,et al.  The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae. , 1988, The Journal of biological chemistry.

[69]  I. Herskowitz.  MAP kinase pathways in yeast: For mating and more , 1995, Cell.

[70]  Frank McCormick,et al.  The GTPase superfamily: conserved structure and molecular mechanism , 1991, Nature.

[71]  A. Varshavsky,et al.  Degradation of G alpha by the N-end rule pathway. , 1994, Science.

[72]  J. Thorner,et al.  Functional domains of a peptide hormone receptor: the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. , 1988, Cold Spring Harbor Symposia on Quantitative Biology.

[73]  J. Thorner,et al.  Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[74]  K. Arai,et al.  Role of SGP2, a suppressor of a gpa1 mutation, in the mating-factor signaling pathway of Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[75]  P. O’Connell,et al.  The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21 , 1990, Cell.

[76]  S. Arkinstall,et al.  Yeast alpha-mating factor receptor and G-protein-linked adenylyl cyclase inhibition requires RAS2 and GPA2 activities. , 1992, Biochemical and biophysical research communications.

[77]  D D Jenness,et al.  Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor. , 1994, Molecular and cellular biology.

[78]  David Botstein,et al.  Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase , 1982, Cell.

[79]  K. Blumer,et al.  Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family , 1996, Nature.

[80]  S. Rogers,et al.  Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. , 1986, Science.

[81]  J. Thorner,et al.  Beta and gamma subunits of a yeast guanine nucleotide-binding protein are not essential for membrane association of the alpha subunit but are required for receptor coupling. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[82]  R. Dixon,et al.  A C‐terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. , 1989, The EMBO journal.

[83]  S. Reed,et al.  Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway , 1995, Nature.

[84]  I. Herskowitz,et al.  From membrane to nucleus: the pathway of signal transduction in yeast and its genetic control. , 1988, Cold Spring Harbor symposia on quantitative biology.

[85]  G. A. Martin,et al.  Molecular cloning of two types of GAP complementary DNA from human placenta. , 1988, Science.

[86]  F. Eckstein,et al.  Determination of the turn-off reaction for the hormone-activated adenylate cyclase. , 1979, The Journal of biological chemistry.

[87]  J. Thorner,et al.  Receptor-G protein signaling in yeast. , 1991, Annual review of physiology.

[88]  M. Wigler,et al.  The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins , 1990, Cell.

[89]  M. Nakafuku,et al.  S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein , 1990, Cell.

[90]  A. Gilman,et al.  Purification of Recombinant G Proteins from Sf9 Cells by Hexahistidine Tagging of Associated Subunits , 1995, The Journal of Biological Chemistry.

[91]  K. Simons,et al.  Regulation of apical transport in epithelial cells by a Gsclass of heterotrimeric G protein , 1993, Nature.

[92]  K. Arai,et al.  GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae , 1989, Molecular and cellular biology.

[93]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[94]  G K Lewis,et al.  Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product , 1985, Molecular and cellular biology.

[95]  Bee-Na Lee,et al.  Overexpression of fIbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brIA and premature initiation of development , 1994, Molecular microbiology.

[96]  E. W. Jones.  Tackling the protease problem in Saccharomyces cerevisiae. , 1991, Methods in enzymology.

[97]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.

[98]  A. Myers,et al.  High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. , 1991, Methods in enzymology.

[99]  G. Natsoulis,et al.  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. , 1987, Methods in enzymology.

[100]  R. W. Davis,et al.  High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[101]  S. Bouvier,et al.  Constitutive mutants in the yeast pheromone response: Ordered function of the gene products , 1989, Cell.

[102]  F. Cross,et al.  Signal transduction in the budding yeast Saccharomyces cerevisiae. , 1994, Current opinion in cell biology.

[103]  B. Wilson,et al.  Cellular variations in heterotrimeric G protein localization and expression in rat pituitary. , 1994, Endocrinology.

[104]  K. Blumer,et al.  Disruption of receptor-G protein coupling in yeast promotes the function of an SST2-dependent adaptation pathway. , 1993, The Journal of biological chemistry.

[105]  J. Thorner,et al.  Mutational activation of the STE5 gene product bypasses the requirement for G protein beta and gamma subunits in the yeast pheromone response pathway , 1994, Molecular and cellular biology.

[106]  H. Bourne,et al.  Separate GTP binding and GTPase activating domains of a G alpha subunit. , 1993, Science.

[107]  D. Jenness,et al.  Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae , 1989, Molecular and cellular biology.

[108]  K. Redding,et al.  Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae , 1991, The Journal of cell biology.

[109]  S. Arkinstall,et al.  Yeast α‐matching factor receptor‐linked G‐protein signal transduction suppresses Ras‐dependent activity , 1991 .

[110]  H. Bourne,et al.  GTPase inhibiting mutations activate the α chain of Gs and stimulate adenylyl cyclase in human pituitary tumours , 1989, Nature.

[111]  M. Whiteway,et al.  Site-directed mutations altering the CAAX box of Ste18, the yeast pheromone-response pathway G gamma subunit. , 1994, Genetics.

[112]  B. Errede,et al.  The proliferation of MAP kinase signaling pathways in yeast. , 1995, Current opinion in cell biology.

[113]  K. Arai,et al.  Suppressors of a gpa1 mutation cause sterility in Saccharomyces cerevisiae. , 1988, Genetics.

[114]  H. Bourne,et al.  Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase. , 1989, The Journal of biological chemistry.

[115]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[116]  L. Hartwell.  Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone , 1980, The Journal of cell biology.

[117]  J. Thorner,et al.  The carboxy-terminal segment of the yeast α-factor receptor is a regulatory domain , 1988, Cell.

[118]  Y. Zheng,et al.  Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. , 1994, The Journal of biological chemistry.