Effects of expression of mammalian G alpha and hybrid mammalian-yeast G alpha proteins on the yeast pheromone response signal transduction pathway.

Scg1, the product of the Saccharomyces cerevisiae SCG1 (also called GPA1) gene, is homologous to the alpha subunits of G proteins involved in signal transduction in mammalian cells. Scg1 negatively controls the pheromone response pathway in haploid cells. Either pheromonal activation or an scg1 null mutation relieves the negative control and leads to an arrest of cell growth in the G1 phase of the cell cycle. Expression of rat G alpha s was previously shown to complement the growth defect of scg1 null mutants while not allowing mating. We have extended this analysis to examine the effects of the short form of G alpha s (which lacks 15 amino acids present in the long form), G alpha i2, G alpha o, and Scg1-mammalian G alpha hybrids. In addition, we have found that constructs able to complement scg1 are also able to inhibit the response to pheromone and mating when expressed in a wild-type SCG1 strain. Overexpression of Scg1 has a similar inhibitory effect. These results are consistent with a model proposed for the action of Scg1 as the alpha component of a heterotrimeric G protein in which the beta gamma component (Ste4/Ste18) activates the pheromone response after dissociation from Scg1. They suggest that the G alpha constructs able to complement scg1 can interact with beta gamma to prevent activation of the pathway but are unable to interact with pheromone receptors to activate the pathway.     

Constitutive mutants in the yeast pheromone response: ordered function of the gene products

The alpha factor pheromone inhibits the division of yeast a cells. A general method was developed for isolating mutants that exhibit constitutive activation of the pheromone response pathway. A dominant allele of the STE4 locus was recovered in addition to recessive mutations in the SCG1 gene. SCG1 and STE4 are known to encode G alpha and G beta homologs, respectively. Analysis of double mutants suggests that the STE4 gene product functions after the SCG1 product but before the STE5 product.     

Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae.

alpha-Factor pheromone inhibits division of yeast a cells. After prolonged exposure to alpha-factor, the cells adapt to the stimulus and resume cell division. The sst2 mutation is known to inhibit adaptation. This report examines adaptation in scg1 (also designated gpa1) and STE4Hpl (Hpl indicates haploid lethal) mutants that exhibit constitutive activation of the pheromone response pathway. Recovery of the STE4Hpl mutant was blocked by the sst2-1 mutation, whereas recovery of the scg1-7 mutant was not completely blocked by sst2-1. These results indicate that both SST2-dependent and -independent mechanisms regulate postreceptor events in the pheromone response pathway. Down regulation of receptors in response to alpha-factor was independent of the signal that was generated in the scg1 mutant.     

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.     

Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.

The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein). This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.     

A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae

MATa cells carrying an sst2 mutation are unable to recover from the G1-specific cell cycle arrest induced by the mating pheromone alpha factor. The KSS1 gene, when overexpressed, suppresses this adaptation defect. KSS1 overexpression also suppresses the recovery defect manifested by cells expressing an alpha factor receptor lacking its 136 amino acid cytoplasmic tail. Because SST2 product and the receptor tail contribute independently to events that allow recovery from pheromone-induced growth arrest, KSS1 function defines a third independent process that promotes desensitization. The KSS1 gene encodes an apparent protein kinase homologous to the CDC28 (S. cerevisiae) and cdc2+ (S. pombe) gene products. The recovery-promoting activity of the KSS1 gene requires a functional WHI1 gene, which encodes a yeast homolog to animal cyclins, suggesting that the KSS1 and WHI1 proteins act in the same growth control pathway.     

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.     

Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by alpha factor pheromone. When sst1 mutants were mixed with normal SST+ cells, the entire population recovered together from alpha factor arrest, suggesting that SST+ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a "barrier" to the diffusion of alpha factor, were lesions in the same genes. These findings suggest that sst1 mutants, are defective in recovery from alpha factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST+ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to alpha factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of alpha factor for a much longer time than SST+ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of alpha factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective ("sterile").     

Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae

MAT alpha haploids with mutations in the STE13 or KEX2 gene, and MATa haploids with mutations in the STE6 or STE14 gene, do not mate with wild-type cells of the opposite mating type. We found that such mutants were able to mate with partners that carry mutations (sst1 and sst2) that cause cells to be supersensitive to yeast mating pheromone action. Mating ability of MAT alpha ste13 and MAT alpha kex2 mutants could also be restored by adding normal MAT alpha cells to mating mixtures or by adding just the appropriate purified pheromone (alpha-factor). Therefore, the mating deficiencies caused by the ste13 and kex2 lesions, and by inference, the ste6 and ste14 mutations, appear to result only from secretion of an insufficient amount of pheromone or a nonfunctional pheromone.     

Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating.

The SCG1 (GPA1), STE4, and STE18 genes of Saccharomyces cerevisiae encode mating-pathway components whose amino acid sequences are similar to those of the alpha, beta, and gamma subunits, respectively, of mammalian G proteins. Genetic evidence suggests that the STE4 and STE18 gene products interact. The mating defects of a set of ste4 mutants were partially suppressed by the overexpression of STE18, and, moreover, a combination of partially defective ste4 and ste18 alleles created a totally sterile phenotype, whereas such synthetic sterility was not observed when the ste18 allele was combined with a weakly sterile ste11 allele. Others have provided genetic evidence consistent with an interaction between the SCG1 (GPA1) and STE4 gene products. We have examined the physical interactions of these subunits by using an in vivo protein association assay. The STE4 and STE18 gene products associated with each other, and this association was disrupted by a mutation in the STE4 gene product whose phenotype was partially suppressed by overexpression of STE18. The STE4 and SCG1 (GPA1) gene products also interacted in the assay, whereas we detected no association of the SCG1 (GPA1) and STE18 gene products.     

a1 protein alters the DNA binding specificity of alpha 2 repressor

The alpha 2 protein of S. cerevisiae, the product of the MAT alpha 2 gene, represses a set of cell-type-specific genes (the a-specific genes) by binding to an operator sequence upstream of each gene. We demonstrate that a second yeast regulatory protein, a1, the product of the MATa1 gene, can alter the binding specificity of alpha 2 so that it no longer recognizes the a-specific gene operator, but instead acquires the ability to recognize a different operator sequence found upstream of haploid-specific genes. Thus, under the influence of a1, alpha 2 can repress haploid-specific genes. An alpha cell expresses alpha 2 but not a1, so that alpha 2 turns off only the a-specific genes. An a/alpha cell makes both a1 and alpha 2, in a ratio that ensures that alpha 2 is distributed between two distinct binding modes: the alpha 2 binding mode and the a1-alpha 2 binding mode. Thus in an a/alpha cell, alpha 2 represses two distinct classes of genes.     

Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae.

Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation. Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells. We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene. Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS). Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis. Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes. Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes. In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1. However, the URS1 mutations prevent any further stimulation of UAS activity by IME1. Repression through URS1 has been shown to require the UME6 gene product. We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product. Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS. We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.     

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.     

sst2 somatostatin receptor mediates cell cycle arrest and induction of p27(Kip1). Evidence for the role of SHP-1.

Activation of the somatostatin receptor sst2 inhibits cell proliferation by a mechanism involving the stimulation of the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory events leading to sst2-mediated growth arrest are not known. Here, we report that treatment of Chinese hamster ovary cells expressing sst2 with the somatostatin analogue, RC-160, led to G1 cell cycle arrest and inhibition of insulin-induced S-phase entry through induction of the cyclin-dependent kinase inhibitor p27(Kip1). Consequently, a decrease of p27(Kip1)-cdk2 association, an inhibition of insulin-induced cyclin E-cdk2 kinase activity, and an accumulation of hypophosphorylated retinoblastoma gene product (Rb) were observed. However, RC-160 had no effect on the p21(Waf1/Cip1). When sst2 was coexpressed with a catalytically inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1 induced entry into cell cycle and down-regulation of p27(Kip1) and prevented modulation by insulin and RC-160 of p27(Kip1) expression, p27(Kip1)-cdk2 association, cyclin E-cdk2 kinase activity, and the phosphorylation state of Rb. In mouse pancreatic acini, RC-160 reverted down-regulation of p27(Kip1) induced by a mitogen, and this effect did not occur in acini from viable motheaten (mev/mev) mice expressing a mutant SHP-1 with markedly deficient enzymes. These findings provide the first evidence that sst2 induces cell cycle arrest through the up-regulation of p27(Kip1) and demonstrate that SHP-1 is required for maintaining high inhibitory levels of p27(Kip1) and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27(Kip1).