Differential transmission of G1 cell cycle arrest and mating signals by Saccharomyces cerevisiae Ste5 mutants in the pheromone pathway.

Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways. G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes. However, the roles of Ste5 in G1 arrest and mating are not fully understood. To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene. Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5M(D248V) and ste5(delta-776)) retained mating capacity. When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating. Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S. cerevisiae. In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.     

Molecular quantification of Saccharomyces cerevisiae α-pheromone secretion

Saccharomyces cerevisiae yeast cells court each other by producing an attractive sex pheromone specific to their mating type. Cells detect the sex pheromone from potential mates using a well-defined intracellular signalling cascade that has become a model for studying signal transduction. In contrast, the factors contributing to the production of pheromone itself are poorly characterized, despite the widespread use of the S.?cerevisiae α-pheromone secretion pathway in industrial fungal protein expression systems. Progress in understanding pheromone secretion has been hindered by a lack of a precise and quantitative pheromone production assay. Here, we present an ELISA-based method for the quantification of α-pheromone secretion. In the absence of pheromone from the opposite mating type, we found that each cell secretes over 550 mature α-pheromone peptides per second; 90% of this total was produced from MF α1. The addition of a-pheromone more than doubled total α-pheromone secretion. This technique offers several improvements on current methods for measuring α-pheromone production and will allow detailed investigation of the factors regulating pheromone production in yeast.     

Simulated diffusion of phosphorylated CheY through the cytoplasm of Escherichia coli

We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in Escherichia coli chemotaxis. A newly developed program, Smoldyn, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S. S. Andrews and D. Bray, Phys. Biol., in press). With Smoldyn, we constructed a three-dimensional model of an E. coli cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli. Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells. The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions. We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell. We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell. The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors. These and other results are left as predictions for future experiments.     

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.     

The PRE and PQ box are functionally distinct yeast pheromone response elements.

Saccharomyces cerevisiae mating pheromones function by binding to cell surface receptors and activating signal transduction processes which regulate gene expression. In this report, we have analyzed the minimum sequence requirements for conferring both a and alpha mating pheromone inducibilities onto a heterologous promoter. Here we show that the repetitive pheromone response element (PRE) which binds to STE12 protein is sufficient to confer pheromone responsiveness only when present in multiple copies. Moreover, by itself, it is preferentially responsive to alpha factor in a cells. In contrast, a single copy of the PQ box of the STE3 upstream activation sequence (UAS) is sufficient to confer a-factor responsiveness in alpha cells. The PQ box binds both MCM1 and MAT alpha 1 in a cooperative manner, and neither the P nor Q site alone is sufficient to confer a-factor responsiveness. In a cells, however, even multiple copies of the PQ box fail to confer alpha-factor responsiveness. Therefore, the PRE and the PQ box are functionally distinct pheromone-responsive elements with opposite cell type specificities. Moreover, these results indicate that the MCM1 protein functions in a signal transduction pathway in a manner analogous to that of its mammalian homolog, the serum response factor, which regulates the expression of the c-fos proto-oncogene in mammals.     

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.     

Methylation is involved in the Ustilago maydis mating response

Methionine auxotrophs of Ustilago maydis were deficient in mating; unlike wild-type cells, they neither induced nor produced normal mating filaments in the presence of compatible cells. The deficiency was most severe when cells were located some distance apart, but when in direct contact with compatible cells methionine auxotrophs mated and infected plants fairly normally. The mating deficiency was genetically linked to the methionine auxotrophy, segregating with it through in planta crosses. Wild-type cells exposed to the methyltransferase inhibitors ethionine and homocysteine thiolactone were similarly impaired in mating. Exogenous methionine, S-adenosylmethionine (SAM), synthetic mating pheromone, or cAMP all compensated for the mating impairment of the auxotrophs to some extent. Although SAM-dependent methylation could influence activities of various molecules in diverse pathways, these observations indicate that the most likely cause of the mating deficiency in met(-) cells is failure to methylate a component of the U. maydis pheromone signal transduction pathway.     

蛾类性信息素生物合成途径及其调控

蛾类通过产生和识别物种特异性性信息素来引发后续交配行为,因此它在两性交配行为中至关重要.它们具有不同碳链长度、末端官能团、不同双键位置和构型等化学结构特征,本文详细讨论了不同蛾类性信息素的合成途径以及催化每一步反应的相关酶系,列举了15种夜蛾科不同亚科常见物种的性信息素组分及其比例,总结了产生特定比例性信息素的可能原因,查阅了夜蛾科不同物种已经鉴别的性信息素,并按照不同亚科、不同官能团和碳链长度对其进行分类,归纳了同一物种及其亲缘物种性信息素组分和比例的变异,总结了产生变异的分子机理,讨论了性信息素变异和物种进化的关系.最后以生物合成激活神经肽(PBAN)为主, 介绍了其调控途径和机制.本文旨在以不同的蛾类性信息素合成途径为线索,从共有合成途径出发深入了解其规律和共性,从特异合成途径出发探究物种间的进化和变异,展望未来的研究方向及其应用.     

Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling.

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.     

The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene.

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.     

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.     

Scp160-dependent mRNA trafficking mediates pheromone gradient sensing and chemotropism in yeast

mRNAs encoding polarity and secretion factors (POLs) target the incipient bud site in yeast for localized translation during division. In pheromone-treated cells we now find that these mRNAs are also localized to the yeast-mating projection (shmoo) tip. However, in contrast to the budding program, neither the She2 nor She3 proteins are involved. Instead, the Scp160 RNA-binding protein binds POL and mating pathway mRNAs and regulates their spatial distribution in a Myo4- and cortical ER-dependent fashion. RNA binding by Scp160 is stimulated by activation of Gpa1, the G protein α subunit regulated by the pheromone receptor, and is required for pheromone gradient sensing, as well as subsequent chemotropic growth and cell-cell mating. These effects are incurred independently of obvious changes in translation; thus, mRNA trafficking is required for chemotropism and completion of the mating program. This is, to our knowledge, the first demonstration of ligand-activated RNA targeting in the development of a simple eukaryote.     

Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.