Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone.

Strains of both haploid mating types containing sst2 mutations are altered in response to pheromone; MATa sst2 cells are supersensitive to alpha-factor, and MAT alpha sst2 cells are supersensitive to a-factor. This phenotype suggests that SST2 encodes a component of the pheromone response pathway that is common to both mating types. We have cloned the SST2 gene by isolation of multicopy plasmids that complement the sst2-1 mutation. One such plasmid contained a 4.5-kilobase HindIII fragment that was able to complement the sst2-1 mutation in high or low copy number, integrated at the SST2 locus, and resulted in an sst2 phenotype when disrupted, indicating that this fragment contained the SST2 gene. We identified the functional region of the complementing DNA fragment by transposon mutagenesis. Sequencing of this fragment identified an open reading frame encoding 698 amino acids at a position that correlated well with the functional region. Expression of an Sst2-beta-galactosidase fusion was haploid specific and induced by exposure to pheromone. We discuss a model in which induction of the SST2 product results in inhibition of a component of the pheromone response pathway, resulting in desensitization to pheromone.

[1]  F. Heffron,et al.  Shuttle Mutagenesis: A Method of Introducing Transposons into Transformable Organisms , 1986 .

[2]  D. O’Day,et al.  Sexual Interactions in Eukaryotic Microbes , 1982 .

[3]  V. Mackay,et al.  Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. I. Isolation and phenotypic characterization of nonmating mutants. , 1974, Genetics.

[4]  T. Manney,et al.  Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein , 1983, Journal of bacteriology.

[5]  H. Riezman,et al.  Two yeast mutants defective in endocytosis are defective in pheromone response , 1986, Cell.

[6]  R. Dale,et al.  A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. , 1985, Plasmid.

[7]  Cloning regulated yeast genes from a pool of lacZ fusions. , 1983, Methods in enzymology.

[8]  S. W. Hall,et al.  Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Tanaka,et al.  Degradation of mating factor by alpha-mating type cells of Saccharomyces cerevisiae. , 1977, Journal of biochemistry.

[10]  J. Thorner,et al.  Yeast mating pheromone alpha factor inhibits adenylate cyclase. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J M Claverie,et al.  PseqIP: A nonredundant and exhaustive protein sequence data bank generated from 4 major existing collections , 1986, Proteins.

[12]  G. Fink,et al.  Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein , 1987, Molecular and cellular biology.

[13]  K. Arai,et al.  Nucleotide sequences of STE2 and STE3, cell type‐specific sterile genes from Saccharomyces cerevisiae , 1985, The EMBO journal.

[14]  W. Duntze,et al.  Mating-factor-mediated sexual agglutination in Saccharomyces cerevisiae , 1978 .

[15]  G. Sprague,,et al.  Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Thorner Pheromonal Regulation of Development in Saccharomyces cerevisiae , 1981 .

[17]  G. Saari,et al.  Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae , 1986, Molecular and cellular biology.

[18]  Leland H. Hartwell,et al.  The yeast α-factor receptor: structural properties deduced from the sequence of the STE2 gene , 1985 .

[19]  V. Mackay,et al.  Induction of yeast mating pheromone a-factor by α cells , 1983, Nature.

[20]  L. Hartwell,et al.  Binding of alpha-factor pheromone to Saccharomyces cerevisiae a cells: dissociation constant and number of binding sites , 1986, Molecular and cellular biology.

[21]  H. Bourne,et al.  A guanine nucleotide-sensitive adenylate cyclase in the yeast Saccharomyces cerevisiae. , 1983, The Journal of biological chemistry.

[22]  T. Ikemura Codon usage and tRNA content in unicellular and multicellular organisms. , 1985, Molecular biology and evolution.

[23]  E. Chen,et al.  Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[24]  G. Sprague,,et al.  Induction of the yeast α-specific STE3 gene by the peptide pheromone a-factor , 1984 .

[25]  S. A. Parent,et al.  Vector systems for the expression, analysis and cloning of DNA sequence in S. cerevisiae , 1985, Yeast.

[26]  P. Lipke,et al.  Sexual agglutination in Saccharomyces cerevisiae , 1981, Journal of bacteriology.

[27]  T. Manney,et al.  Mutants of SACCHAROMYCES CEREVISIAE Resistant to the α Mating-Type Factor , 1976 .

[28]  R. E. Esposito,et al.  Genetic map of Saccharomyces cerevisiae, edition 9. , 1985, Microbiological reviews.

[29]  A. Gilman G proteins and dual control of adenylate cyclase , 1984, Cell.

[30]  J. Thorner,et al.  The yeast repeated element sigma contains a hormone-inducible promoter , 1987, Molecular and cellular biology.

[31]  W. H. Mager,et al.  A comparison of yeast ribosomal protein gene DNA sequences. , 1984, Nucleic acids research.

[32]  B. Goldman,et al.  Saccharomyces cerevisiae Mutants Unresponsive tocL-Factor Pheromone: Ca-Factor Binding andExtragenic Suppression , 1987 .

[33]  W. Duntze,et al.  2 – The Isolation, Characterization, and Physiological Effects of the Saccharomyces cerevisiae Sex Pheromones , 1981 .

[34]  I. Herskowitz,et al.  Structure of a yeast pheromone gene (MFα): A putative α-factor precursor contains four tandem copies of mature α-factor , 1982, Cell.

[35]  R. Rothstein One-step gene disruption in yeast. , 1983, Methods in enzymology.

[36]  A. Myers,et al.  Yeast/E. coli shuttle vectors with multiple unique restriction sites , 1986, Yeast.

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

[38]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

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

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

[41]  J. Broach,et al.  Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. , 1979, Gene.

[42]  J W Szostak,et al.  Yeast transformation: a model system for the study of recombination. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[43]  G. Sprague,,et al.  Yeast peptide pheromones, a-factor and α-factor, activate a common response mechanism in their target cells , 1986, Cell.

[44]  Leland H. Hartwell,et al.  Binding of α-factor pheromone to yeast a cells: Chemical and genetic evidence for an α-factor receptor , 1983, Cell.

[45]  I. Herskowitz,et al.  Control of yeast cell type by the mating type locus. I. Identification and control of expression of the a-specific gene BAR1. , 1981, Journal of molecular biology.

[46]  K. Nasmyth,et al.  The structure of transposable yeast mating type loci , 1980, Cell.

[47]  E. Southern Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[48]  J. Kurjan,et al.  Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating , 1985, Molecular and cellular biology.

[49]  K. Nasmyth,et al.  Identification and comparison of two sequence elements that confer cell-type specific transcription in yeast , 1985, Nature.

[50]  D. Sibley,et al.  Regulation of transmembrane signaling by receptor phosphorylation , 1987, Cell.

[51]  J. Messing New M13 vectors for cloning. , 1983, Methods in enzymology.

[52]  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.

[53]  L. Stryer,et al.  G proteins: a family of signal transducers. , 1986, Annual review of cell biology.

[54]  L. Stryer,et al.  Cyclic GMP cascade of vision. , 1986, Annual review of neuroscience.

[55]  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.

[56]  J. Bennetzen,et al.  Codon selection in yeast. , 1982, The Journal of biological chemistry.