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PubMed1997

The G beta gamma complex of the yeast pheromone response pathway. Subcellular fractionation and protein-protein interactions.

Genetic evidence suggests that the yeast STE4 and STE18 genes encode G beta and G gamma subunits, respectively, that the G betagamma complex plays a positive role in the pheromone response pathway, and that its activity is subject to negative regulation by the G alpha subunit (product of the GPA1 gene) and to positive regulation by cell-surface pheromone receptors. However, as yet there is no direct biochemical evidence for a G betagamma protein complex associated with the plasma membrane. We found that the products of the STE4 and STE18 genes are stably associated with plasma membrane as well as with internal membranes and that 30% of the protein pool is not tightly associated with either membrane fraction. A slower-migrating, presumably phosphorylated, form of Ste4p is enriched in the non-membrane fraction. The Ste4p and Ste18p proteins that had been extracted from plasma membranes with detergent were found to co-sediment as an 8 S particle under low salt conditions and as a 6 S particle in the presence of 0.25 M NaCl; the Ste18p in these fractions was precipitated with anti-Ste4p antiserum. Under the conditions of our assay, Gpa1p was not associated with either particle. The levels of Ste4p and Ste18p accumulation in mutant cells provided additional evidence for a G betagamma complex. Ste18p failed to accumulate in ste4 mutant cells, and Ste4p showed reduced levels of accumulation and an increased rate of turnover in ste18 mutant cells. The gpa1 mutant blocked stable association of Ste4p with the plasma membrane, and the ste18 mutant blocked stable association of Ste4p with both plasma membranes and internal membranes. The membrane distribution of Ste4p was unaffected by the ste2 mutation or by down-regulation of the cell-surface receptors. These results indicate that at least 40% of Ste4p and Ste18p are part of a G betagamma complex at the plasma membrane and that stable association of this complex with the plasma membrane requires the presence of G alpha.

Cell Compartmentation↗
PubMed1988

The a-factor pheromone of Saccharomyces cerevisiae is essential for mating.

The Saccharomyces cerevisiae pheromone a-factor is produced by a cells and interacts with alpha cells to cause cell cycle arrest and other physiological responses associated with mating. Two a-factor structural genes, MFA1 and MFA2, have been previously cloned with synthetic probes based on the a-factor amino acid sequence (A. Brake, C. Brenner, R. Najarian, P. Laybourn, and J. Merryweather, cited in M.-J. Gething [ed.], Protein transport and secretion, 1985). We have examined the function of these genes in a-factor production and mating by construction and analysis of chromosomal null mutations. mfa1 and mfa2 single mutants each exhibited approximately half the wild-type level of a-factor activity and were proficient in mating, whereas the mfa1 mfa2 double mutant produced no a-factor and was unable to mate. These results demonstrate that both genes are functional, that each gene makes an equivalent contribution to the a-factor activity and mating capacity of a cells, and that a-factor plays an essential role in mating. Strikingly, exogenous a-factor did not alleviate the mating defect of the double mutant, suggesting that an a cell must be producing a-factor to be an effective mating partner.

Amino Acid Sequence↗
PubMed1989

Fetal, hormonal and experiential factors influencing the mating-induced regulation of infanticide in male house mice.

When a male house mouse encounters a neonate he either attempts to kill it or he does not harm it. An unusual aspect of his response is that adaptive, time-dependent changes in behavior toward pups result from a unique stimulus-response system triggered specifically by ejaculation. In virgin male CF-1 mice, about 50% of all males are spontaneously infanticidal when they encounter a pup while the other 50% are typically "parental." The stimulus of ejaculation causes virtually all males to kill pups; however, by the time offspring are born three weeks after mating, infanticide is inhibited and almost all males now behave parentally toward pups. Our experiments examine fetal, experiential, and hormonal factors influencing the changes in infanticidal and parental behavior that occur in male mice as a result of mating. Males who developed in utero between two female fetuses, and were thus exposed to relatively low testosterone concentrations during fetal development, were significantly more likely to exhibit infanticide--both before and after mating--than were males who developed between two male fetuses. Concurrent exposure to testosterone appears required in order for naive males to exhibit infanticide and for spontaneously parental males to become infanticidal after ejaculation. In contrast, neither testosterone nor pituitary hormones appeared responsible for the timed inhibition of infanticide occurring by three weeks after mating, since castrated and hypophysectomized males showed a response pattern similar to intact males. The mating-induced inhibition phenomenon appears to be a neurally timed and mediated response that operates independently from pituitary hormone secretions or changes in gonadal hormones resulting from mating.

Aggression↗
PubMed1996

Role of type I myosins in receptor-mediated endocytosis in yeast.

Type I myosins are thought to drive actin-dependent membrane motility, but the direct demonstration in vivo of their involvement in specific cellular processes has been difficult. Deletion of the genes MYO3 and MYO5, which encode the yeast type I myosins, almost abolished growth. A double-deleted mutant complemented with a MYO5 temperature-sensitive allele (myo5-1) showed a strong defect in the internalization step of receptor-mediated endocytosis, whereas the secretory pathway remained apparently unaffected. Thus, myosin I activity is required for a budding event in endocytosis but not for several other aspects of membrane traffic.

Alleles↗
PubMed1988

The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae.

The STE2 gene of Saccharomyces cerevisiae encodes a 431-residue protein containing seven hydrophobic segments that is thought to be an essential component of the cell-surface receptor for alpha-factor in MATa haploids. Methods were devised to prepare membrane fractions from MATa cells that retained high levels of alpha-factor binding activity, consistent with the view that the alpha-factor receptor resides in the plasma membrane. To demonstrate that the membrane constituent responsible for alpha-factor binding was the STE2 polypeptide, specific antibodies were generated and used to identify STE2-related polypeptides by radiolabeling, immunoprecipitation, and polyacrylamide gel electrophoresis. Under conditions of complete solubilization, the major form of the STE2 gene product detected was a glycoprotein with an apparent molecular weight of 49,000. Affinity labeling of yeast membrane preparations by chemical cross-linking to 35S-alpha-factor indicated that a molecule of 49,000 molecular weight was the major alpha-factor-binding species. This alpha-factor-binding species was shown to be the product of the STE2 gene in three ways. First, MATa haploids carrying the STE2 gene on a multicopy plasmid overproduced alpha-factor binding activity about 15-fold. Second, MATa cells completely lacking a STE2 gene showed only nonspecific binding of alpha-factor (equivalent to the level displayed by MAT alpha haploids) and possessed no species that could be cross-linked to 35S-alpha-factor. Third, MATa cells expressing a truncated but functional STE2 gene (in which the COOH-terminal 135-hydrophilic residues were deleted) produced a protein detected by cross-linking to 35S-alpha-factor of apparent molecular weight 33,000, close to the size expected for the predicted abbreviated STE2 polypeptide. These findings demonstrate unequivocally that the STE2 gene product is the membrane component responsible for the ligand recognition function of the yeast alpha-factor receptor.

Genes↗
PubMed1987

Alpha-factor inhibition of the rate of cell passage through the "start" step of cell division in Saccharomyces cerevisiae yeast: estimation of the division delay per alpha-factor.receptor complex.

A highly sensitive, kinetically unambiguous assay for alpha-factor-induced delay of cell passage through the "start" step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the alpha-factor execution point of start. The t1/2 for cell passage through start by this population of cells is 31 min in the absence of alpha-factor. The inhibition constant, KI, represents the alpha-factor concentration which produces a 50% inhibition of this rate and is equal to 2 X 10(-10) M. A second assay for maximal cell division arrest by alpha-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125 +/- 5 h at saturating alpha-factor, and a K50 (that is, an alpha-factor concentration which produces a half-maximal response) of 2.5 X 10(-8) M. Both assays were performed in the effective absence of alpha-factor inactivation. Values of the dissociation constant KD and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5 X 10(-8) M and 10(4), respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor.alpha-factor complex which is present on the cell at equilibrium producing a 43 +/- 10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to alpha-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM alpha-factor to a 125 +/- 5 h maximum arrest at saturating alpha-factor concentrations of greater than 170 nM. The possible significance of this observation toward the mechanism of alpha-factor-induced cell division arrest is discussed.

Cell Division↗
PubMed1988

The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone.

STE2 encodes a component of the S. cerevisiae alpha-pheromone receptor that is essential for induction of physiological changes associated with mating. Analysis of C-terminal truncation mutants of STE2 demonstrated that the essential sequences for ligand binding and signal transduction are included within a region containing seven putative transmembrane domains. However, truncation of the C-terminal 105 amino acids of the receptor resulted in a 4- to 5-fold increase in cell-surface pheromone binding sites, a 10-fold increase in pheromone sensitivity, a defect in recovery of cell division after pheromone treatment, and a defect in pheromone-induced morphogenesis. Overproduction of STE2 resulted in about a 6-fold increase in alpha-pheromone binding capacity but did not produce the other phenotypes associated with the ste2-T326 mutant receptor. We conclude that the C-terminus of the receptor is responsible for one aspect of cellular adaptation to pheromone that is distinct from adaptation controlled by the SST2 gene, for decreasing the stability of the receptor, and for some aspect of cellular morphogenesis.

Crosses, Genetic↗
PubMed2001

Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor.

Heterologous expression of the human neurotensin receptor type I (hNT1-R) has been achieved in the yeast Saccharomyces cerevisiae. Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast. High-affinity binding sites for 125I-labeled-[monoiodo-Tyr3]neurotensin ([125I-Tyr3]NT) were detected on hNT1-R-expressing cells with Kd and Bmax values of 3.2 nM and of 500 receptors per cell, respectively. Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT1-R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT1-R expressed in mammalian cells. Interestingly, neurotensin activated the pheromone pathway in hNT1-R-expressing cells in a dose-dependent fashion, as revealed by a beta-galactosidase activity assay with a pheromone-responsive Fus1:lacZ construct. Mutational inactivation of the SST2 and STE2 genes increased the level of beta-galactosidase activity in response to neurotensin by twofold. Recombinant hNT1-R-producing cells, which lacked the endogenous G-protein-coupled receptor for the alpha pheromone, mated with wild-type MATalpha haploid cells in response to neurotensin, leading to bona fide diploid zygote formation. This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist-induced, mating process.

Binding, Competitive↗
PubMed1998

A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus.

Pheromone signaling plays an essential role in the mating and sexual development of mushroom fungi. Multiallelic genes encoding the peptide pheromones and their cognate 7-transmembrane helix (7-TM) receptors are sequestered in the B mating type locus. Here we describe the isolation of the B6 mating type locus of Coprinus cinereus. DNA sequencing and transformation analysis identified nine genes encoding three 7-TM receptors and six peptide pheromone precursors embedded within 17 kb of mating type-specific sequence. The arrangement of the nine genes suggests that there may be three functionally independent subfamilies of genes each comprising two pheromone genes and one receptor gene. None of the nine B6 genes showed detectable homology to corresponding B gene sequences in the genomic DNA from a B3 strain, and each of the B6 genes independently alter B mating specificity when introduced into a B3 host strain. However, only genes in two of the B6 groups were able to activate B-regulated development in a B42 host. Southern blot analysis showed that these genes failed to cross-hybridize to corresponding genes in the B42 host, whereas the three genes of the third subfamily, which could not activate development in the B42 host, did cross-hybridize. We conclude that cross-hybridization identifies the same alleles of a particular subfamily of genes in different B loci and that B6 and B42 share alleles of one subfamily. There are an estimated 79 B mating specificities: we suggest that it is the different allele combinations of gene subfamilies that generate these large numbers.

Amino Acid Sequence↗
PubMed1996

Ste50p sustains mating pheromone-induced signal transduction in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the heterotrimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free G beta gamma then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the alpha-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae.

Alleles↗
PubMed1993

The A mating-type genes of the mushroom Coprinus cinereus are not differentially transcribed in monokaryons and dikaryons.

The A mating type factor of Coprinus cinereus regulates part of a developmental sequence that leads to the conversion of the asexual monokaryon into the fertile dikaryon. The A42 factor is a complex of seven genes, at least four of which are involved in determining the specificity of mating interactions. In this report we show that the A42 genes are constitutively expressed in both monokaryons and dikaryons. This has important implications with respect to intracellular recognition of a compatible mating, which requires an interaction between proteins already present within the cells of the mating partners, and for the subsequent maintenance of dikaryotic growth.

Blotting, Northern↗
PubMed2006

Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade.

Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin of new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus Cryptococcus. Previous studies revealed that two PKA catalytic subunits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the Cryptococcus neoformans var. grubii serotype A lineage compared to divergent C. neoformans var. neoformans serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species Cryptococcus gattii by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens.

Catalytic Domain↗
PubMed1994

Systematic mutagenesis of the yeast mating pheromone receptor third intracellular loop.

Signal transduction in the mating pathway of the yeast Saccharomyces cerevisiae is initiated by binding of a peptide pheromone to a G protein-coupled receptor (Ste2). We systematically have mutated the third intracellular loop of the Ste2 receptor to investigate its functional significance. We substituted each of the 13 amino acids in the loop with alanine individually or together with one other residue. In addition, we used a site-directed random replacement mutagenesis technique to replace a region encoding three amino acids in the loop with random sequence. Over 80 such Ste2 mutants have been analyzed by several functional and biochemical criteria in a yeast strain that carries a genomic deletion of the STE2 gene. The mutant phenotypes range from fully functional to severely compromised in signaling. The observation that amino acid substitutions in the third intracellular loop of the Ste2 receptor can affect activation of the yeast mating response implicates the loop in this signal transduction pathway. The types of mutations that compromise the function of the receptor may provide clues to the physical interaction between the receptor and the G protein.

Amino Acid Sequence↗
PubMed1992

The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus.

The A mating-type factor is one of two gene complexes that allows mating cells of the mushroom Coprinus cinereus to recognize self from nonself and to regulate a pathway of sexual development that leads to meiosis and sporulation. We have identified seven A genes separated into two subcomplexes corresponding to the classical A alpha and A beta loci. Four genes, one alpha and three beta, all coding for proteins with a homeo domain-related motif, determine A-factor specificity; their allelic forms are so different in sequence that they do not cross-hybridize. It requires only one of these four genes to be heteroallelic in a cell to trigger A-regulated sexual development, and it is the different combinations of their alleles that generate the multiple A factors found in nature. The other three genes cause no change in cell morphology and may regulate the activity of the four specificity genes.

Alleles↗