Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.     

Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects

We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.     

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.     

Pheromone-induced phosphorylation of a G protein beta subunit in S. cerevisiae is associated with an adaptive response to mating pheromone

The mating pheromone response in S. cerevisiae is activated by a G protein-mediated signaling pathway in which G beta gamma is the active transducer of the signal. When exogenous pheromone is added to vegetatively growing cells, G beta is rapidly phosphorylated at several sites; phosphorylation does not require de novo protein synthesis. A mutation in G beta was constructed that eliminates signal-induced phosphorylation. This mutation leads to enhanced sensitivity to and impaired ability to recover from pheromone, but does not affect the ability of G beta gamma to transmit the mating signal. These phenotypes suggest that G protein phosphorylation mediates an adaptive response to pheromone-induced signaling. G beta phosphorylation does not require either the pheromone receptor C-terminus or the product of the SST2 gene, both of which mediate separate adaptive responses to pheromone. However, G beta phosphorylation is greatly facilitated by the presence of the G alpha subunit, which has also been shown to participate in an adaptation to pheromone.     

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.     

Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway.

The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn. The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.     

S. cerevisiae alpha pheromone receptors activate a novel signal transduction pathway for mating partner discrimination.

Wild-type S. cerevisiae cells of both mating types prefer partners producing high levels of pheromone and mate very infrequently to cells producing no pheromone. However, some mutants that are supersensitive to pheromone lack this ability to discriminate. In this study, we provide evidence for a novel role of alpha pheromone receptors in mating partner discrimination that is independent of the known G protein-mediated signal transduction pathway. Furthermore, in response to pheromone, receptors become localized to the emerging region of morphogenesis that is positioned adjacent to the nucleus, suggesting that receptor localization may be involved in mating partner discrimination. Actin, myosin 2, and clathrin heavy chain are involved in mating partner discrimination, since strains carrying mutations in the genes encoding these proteins result in a small but significant defect in mating partner discrimination.     

The carboxy-terminal tail of the Ste2 receptor is involved in activation of the G protein in the Saccharomyces cerevisiae alpha-pheromone response pathway

The Ste2 gene encodes the yeast alpha-pheromone receptor that belongs to the superfamily of seven-transmembrane G protein-coupled receptors. Binding of pheromone induces activation of the heterotrimeric G protein triggering growth arrest in G1 phase and induction of genes required for mating. By random PCR-mediated mutagenesis we isolated mutant 8L4, which presents a substitution of an asparagine residue by serine at position 388 of the alpha-factor receptor. The 8L4 mutant strain shows phenotypic defects such as: reduction in growth arrest after pheromone treatment, diminished activation of the Fus1 gene, and impaired mating competence. The asparagine residue lies in the second half of the intracellular protruding C-terminal tail of the receptor, and its replacement by serine affects interaction with both the G(alpha) and Gbeta subunits. Since expression of the receptor as well as its kinetic parameters, i.e., ligand affinity and receptor number, are unaffected in the mutant strain, we propose that association of the C-terminal tail of the receptor with G(alpha) and Gbeta subunits is required for proper activation of the heterotrimeric G protein. Besides its described role in downregulation and in formation of preactivation complex, the results here shown indicate that the C-terminal tail of the receptor plays an active role in transmitting the stimulus of mating pheromone to the heterotrimeric G protein.     

酵母交配过程的极性生长及其调控机制

酿酒酵母单倍体细胞能够与相反交配型的单倍体细胞发生交配。交配时酿酒酵母放弃原有出芽位点,根据信息素的浓度梯度,重新选择生长位点,向相反交配型细胞伸出突起进行极性生长。交配因子受体指导选择交配突起的位点,通过G蛋白激活Ste20p,将信号经由Ste11p、Ste7p和Fus3p组成的MAPK模块传递到Far1p和Ste12p等因子,调控相关基因的转录,抑制原有的出芽位点,选择新的生长位点,并使细胞周期停止在G1期,G蛋白与Cdc24p、Cdc42p和Bem1p等蛋白作用,聚集在细胞,使得肌协蛋白细胞骨架在交配突起处聚集,呈极性化分布,使细胞发生极性生长。     

The yeast G-protein homolog is involved in the mating pheromone signal transduction system.

I have isolated a new type of sterile mutant of Saccharomyces cerevisiae, carrying a single mutant allele, designated dac1, which was mapped near the centromere on chromosome VIII. The dac1 mutation caused specific defects in the pheromone responsiveness of both a and alpha cells and did not seem to be associated with any pleiotropic phenotypes. Thus, in contrast to the ste4, ste5, ste7, ste11, and ste12 mutations, the dac1 mutation had no significant effect on such constitutive functions of haploid cells as pheromone production and alpha-factor destruction. The characteristics of this phenotype suggest that the DAC1 gene encodes a component of the pheromone response pathway common to both a and alpha cells. Introduction of the GPA1 gene encoding an S. cerevisiae homolog of the alpha subunit of mammalian guanine nucleotide-binding regulatory proteins (G proteins) into sterile dac1 mutants resulted in restoration of pheromone responsiveness and mating competence to both a and alpha cells. These results suggest that the dac1 mutation is an allele of the GPA1 gene and thus provide genetic evidence that the yeast G protein homolog is directly involved in the mating pheromone signal transduction pathway.     

Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment.

The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.