Separate roles for N‐ and C‐termini of the STE4 (β) subunit of the Saccharomyces cerevisiae G protein in the mediation of the growth arrest. Lack of growth‐arresting activity of mammalian βγ complexes

Mating pheromone signal transduction in Saccharomyces cerevisiae involves a G protein composed to Scg1p (Gpa1p), Ste4p and Ste18p subunits, homologous to the α, β and γ subunits of mammalian G proteins. Growth arrest in G1 phase is activated by the Ste4p/Ste18p complex via a downstream pathway and it is negatively controlled by the Scg1p subunit. Here we explored whether mammalian β or γ subunits could functionally substitute for their yeast homologues. While no evidence was obtained for functional replacement of Ste4p and Ste18p, we found that overexpression of Ste18p potentiated the effect of hybrid proteins in which the N terminus of the Ste4p subunit was replaced by that of the mammalian β, ste4 mutants having deletions in the N terminus showed a decreased activity in signalling to the downstream effector of the pheromone response. This defect was totally cured by overexpression of Ste18p, indicating that the truncated forms of Ste4p have retained their ability to form an active complex with Ste18p. Removal of six amino acids from the C terminus of Ste4p rendered a completely inactive subunit and this defect persisted in hybrids where the C terminus was placed by that of the β subunit, indicating that the C terminus of Ste4p is essential to trigger the effector of the yeast pheromone response pathway.

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

[2]  L. Birnbaumer,et al.  STE2/SCG1‐dependent inhibition of STE4‐induced growth arrest by mutant STE4ΔC6 in the yeast pheromone response pathway , 1995, FEBS letters.

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

[4]  K. Clark,et al.  Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating , 1993, Molecular and cellular biology.

[5]  M. Simon,et al.  Subunits βγ of heterotrimeric G protein activate β2 isoform of phospholipase C , 1992, Nature.

[6]  M. Camps,et al.  Isozyme-selective stimulation of phospholipase C-β2 by G protein βγ-subunits , 1992, Nature.

[7]  M. Whiteway,et al.  The protein kinase homologue Ste20p is required to link the yeast pheromone response G‐protein beta gamma subunits to downstream signalling components. , 1992, The EMBO journal.

[8]  M. Whiteway,et al.  Dominant‐negative mutants of a yeast G‐protein beta subunit identify two functional regions involved in pheromone signalling. , 1992, The EMBO journal.

[9]  G. Schultz,et al.  Different β-subunits determine G-protein interaction with transmembrane receptors , 1992, Nature.

[10]  H. Ploegh,et al.  The WD‐40 repeat , 1992, FEBS letters.

[11]  E. Neer,et al.  Specificity of G protein beta and gamma subunit interactions. , 1992, The Journal of biological chemistry.

[12]  N. Gautam,et al.  Interaction between G-protein beta and gamma subunit types is selective. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. Gilman,et al.  Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits. , 1991, Science.

[14]  G. Sprague,,et al.  Signal transduction in yeast mating: receptors, transcription factors, and the kinase connection. , 1991, Trends in genetics : TIG.

[15]  L. Birnbaumer,et al.  Gamma-subunits of G proteins, but not their alpha- or beta-subunits, are polyisoprenylated. Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates. , 1991, The Journal of biological chemistry.

[16]  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.

[17]  H. Khorana,et al.  Sites of interaction in the complex between beta- and gamma-subunits of transducin. , 1990, The Journal of biological chemistry.

[18]  Stanley Fields,et al.  Phermone response in yeast , 1990 .

[19]  Y. Nabeshima,et al.  Regulation of the chicken embryonic myosin light-chain (L23) gene: existence of a common regulatory element shared by myosin alkali light-chain genes , 1990, Molecular and cellular biology.

[20]  Y. Kang,et al.  Effects of expression of mammalian G alpha and hybrid mammalian-yeast G alpha proteins on the yeast pheromone response signal transduction pathway , 1990, Molecular and cellular biology.

[21]  R. Plasterk,et al.  Characterization of a G-protein beta-subunit gene from the nematode Caenorhabditis elegans. , 1990, Journal of molecular biology.

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

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

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

[25]  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.

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

[27]  J. Hurley,et al.  Cloning of a Drosophila melanogaster guanine nucleotide regulatory protein beta-subunit gene and characterization of its expression during development. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[28]  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.

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

[30]  M. Nakafuku,et al.  Occurrence in Saccharomyces cerevisiae of a gene homologous to the cDNA coding for the alpha subunit of mammalian G proteins. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[31]  D. Ecker,et al.  Chemical synthesis and expression of a cassette adapted ubiquitin gene. , 1987, The Journal of biological chemistry.

[32]  D. Stengel,et al.  β‐Subunits of the human liver Gs/Gi signal‐transducing proteins and those of bovine retinal rod cell transducin are identical , 1986, FEBS letters.

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

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

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

[36]  N. Ryba,et al.  Sequence of the beta-subunit of the phosphatidylinositol-specific phospholipase C-directed GTP-binding protein from squid (Loligo forbesi) photoreceptors. , 1991, Biochemical Journal.

[37]  S. Fields Pheromone response in yeast. , 1990, Trends in biochemical sciences.

[38]  R. Plasterk,et al.  Characterization of a G-protein alpha-subunit gene from the nematode Caenorhabditis elegans. , 1990, Journal of Molecular Biology.

[39]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[40]  D. Tipper,et al.  Effects of Expression of Mammalian GeL and Hybrid Mammalian-Yeast Ga Proteins on the Yeast Pheromone Response Signal Transduction Pathway , 2022 .

[41]  K. Clark,et al.  Genetic Identification of Residues Involved in Association of oa and a G-Protein Subunitst , 2022 .