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At least 451 records · Page 25Linked to original sources
PubMed1989

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.

Adaptation, Physiological↗
PubMed1996

Regulation of the G-protein-coupled alpha-factor pheromone receptor by phosphorylation.

The alpha-factor pheromone receptor activates a G protein signaling cascade that stimulates MATa yeast cells to undergo conjugation. The cytoplasmic C terminus of the receptor is not necessary for G protein activation but instead acts as a regulatory domain that promotes adaptation to alpha-factor. The role of phosphorylation in regulating the alpha-factor receptor was examined by mutating potential phosphorylation sites. Mutation of the four most distal serine and threonine residues in the receptor C terminus to alanine caused increased sensitivity to alpha-factor and a delay in recovering from a pulse of alpha-factor. 32PO4 labeling experiments demonstrated that the alanine substitution mutations decreased the in vivo phosphorylation of the receptor. Phosphorylation apparently alters the regulation of G protein activation, since neither receptor number nor affinity for ligand was significantly altered by mutation of the distal phosphorylation sites. Furthermore, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased sensitivity to alpha-factor, which suggests that regulation by phosphorylation can occur at the cell surface and is independent of endocytosis. Mutation of the distal serine and threonine residues of the receptor also caused a slight defect in alpha-factor-induced morphogenesis, but the defect was not as severe as the morphogenesis defect caused by truncation of the cytoplasmic C terminus of the receptor. These distal residues in the C terminus play a special role in receptor regulation, since mutation of the next five adjacent serine and threonine residues to alanine did not affect the sensitivity to alpha-factor. Altogether, these results indicate that phosphorylation plays an important role in regulating alpha-factor receptor function.

Binding Sites↗
PubMed1995

Novel antagonist to agonist switch in chimeric G protein-coupled alpha-factor peptide receptors.

The alpha-factor analog desTrp1,Ala3 alpha-factor (dTA-alpha-factor) acts as an antagonist for both the S. cerevisiae alpha-factor receptor (c-alpha-FR) and the S. kluyveri alpha-factor receptor (k-alpha FR). Chimeric alpha-factor receptors in which a portion (residues 250-303) of the seven hydrophobic segments were derived from the k-alpha FR and the rest from c-alpha FR conferred potent activation of cellular responses by dTA-alpha-factor including induction of FUS1 expression without significantly affecting binding. Chimeric receptors with all hydrophobic segments derived from one receptor did not express sensitivity. We propose that dTA-alpha-factor represents an antagonist that that has an activation energy barrier to activating c-alpha FR and k-alpha FR but which retains a latent potential to act as an agonist.

Binding, Competitive↗
PubMed2003

The C-terminal tail preceding the CAAX box of a yeast G protein gamma subunit is dispensable for receptor-mediated G protein activation in vivo.

The gamma subunits of heterotrimeric G proteins are required for receptor-G protein coupling. The C-terminal domains of Ggamma subunits can contact receptors and influence the efficiency of receptor-G protein coupling in vitro. However, it is unknown whether receptor interaction with the C terminus of Ggamma is required for signaling in vivo. To address this question, we cloned Ggamma homologs with diverged C-terminal sequences from five species of budding yeast. Each Ggamma homolog functionally replaced the Ggamma subunit of the yeast Saccharomyces cerevisiae (STE18 gene product). Mutagenesis of S. cerevisiae Ste18 likewise indicated that specific C-terminal sequence motifs are not required for signaling. Strikingly, an internal in-frame deletion removing sequences preceding the C-terminal CAAX box of Ste18 did not impair signaling by either of its cognate G protein-coupled pheromone receptors. Therefore, receptor interaction with the C-terminal domain of yeast Ggamma is not required for receptor-mediated G protein activation in vivo. Because the mechanism of G protein activation by receptors is conserved from yeast to humans, mammalian receptors may not require interaction with the tail of Ggamma for G protein activation in vivo. However, receptor-Ggamma interaction may modulate the efficiency of receptor-G protein coupling or promote activation of Gbetagamma effectors that co-cluster with receptors.

GTP-Binding Protein gamma Subunits↗
PubMed2003

Alpha-pheromone-induced "shmooing" and gene regulation require white-opaque switching during Candida albicans mating.

A 14-mer alpha-pheromone peptide of Candida albicans was chemically synthesized and used to analyze the role of white-opaque switching in the mating process. The alpha-pheromone peptide blocked cell multiplication and induced "shmooing" in a/a cells expressing the opaque-phase phenotype but not in a/a cells expressing the white-phase phenotype. The alpha-pheromone peptide induced these effects at 25 degrees C but not at 37 degrees C. An analysis of mating-associated gene expression revealed several categories of gene regulation, including (i) MTL-homozygous-specific, pheromone stimulated, switching-independent (CAG1 and STE4); (ii) mating type-specific, pheromone-induced, switching-independent (STE2); and (iii) pheromone-induced, switching-dependent (FIG1, KAR4, and HWP1). An analysis of switching-regulated genes revealed an additional category of opaque-phase-specific genes that are downregulated by alpha-pheromone only in a/a cells (OP4, SAP1, and SAP3). These results demonstrate that alpha-pheromone causes shmooing, the initial step in the mating process, only in a/a cells expressing the opaque phenotype and only at temperatures below that in the human host. These results further demonstrate that although some mating-associated genes are stimulated by the alpha-pheromone peptide in both white- and opaque-phase cells, others are stimulated only in opaque-phase cells, revealing a category of gene regulation unique to C. albicans in which alpha-pheromone induction requires the white-opaque transition. These results demonstrate that in C. albicans, the mating process and associated gene regulation must be examined within the context of white-opaque switching.

ATP-Binding Cassette Transporters↗
PubMed1978

Arousal, personality, and assortative mating in marriage: generalizability and cross-cultural factors.

The significant assortative mating for the sensation-seeking motive in (married) American students reported by Farley and Davis was significantly cross validated on American (N = 160) and German (N = 160) samples randomly selected from two comparable cities in the Federal German Republic and the United States. A control condition of age-matched unmarried pairs was included. Spouses were found to have 19% and 29% of sensation-seeking variance in common in the American and German samples, respectively. The later figure is exceptionally high for the literature on assortative mating (exceeding or approximating some of the relationships found for intellectual measures). No significant variance was in common for the unmarried pairs. When a simple and easily measured construct with such wide implications as the sensation-seeking motive can be shown to be so significantly involved in marital partner similarity, then its consequences for successful and unsuccessful marriage should be pursued with vigor. Such a program in research and therapy is recommended and a theoretical model suggested.

Adolescent↗
PubMed1989

rRNA transcription initiation is decreased by inhibitors of the yeast cell cycle control step "start".

Inhibitors of the "start" regulatory step in the cell cycle of the yeast Saccharomyces cerevisiae are known by indirect studies to perturb RNA metabolism. We have investigated these effects further and show here by a pulse-labeling and quantitative hybridization procedure that pre-rRNA transcription was substantially decreased by five inhibitors of start but was transiently stimulated by the mating pheromone alpha-factor. Thus in contrast to the effects of the other start inhibitors, the inhibition of start by alpha-factor is unrelated to this aspect of biosynthetic activity. Mating factor treatment also stimulated the synthesis rate of poly(A)+ RNA. The start inhibitors o-phenanthroline and L-ethionine inhibited pre-rRNA transcription with little effect on poly(A)+ RNA synthesis rates. Northern analysis showed that all inhibitors of start also inhibited pre-rRNA transcript cleavage, a process that has been dissociated from the inhibition of start. Most inhibitors also affected ATP pool size. One inhibitor, o-phenanthroline, markedly induced the general control response.

Adenosine Triphosphate↗
PubMed1996

Defective threonine-linked glycosylation of human insulin-like growth factor in mutants of the yeast Saccharomyces cerevisiae.

Mutants of the yeast Saccharomyces cerevisiae were identified, in which O-glycosylation at threonine 29 of a heterologous protein, human insulin-like growth factor (hIGF-1), is defective. In mutant M195, O-glycosylation of hIGF-1, but not of yeast proteins chitinase and a-agglutinin, was reduced; in mutant M577 yeast proteins were affected besides hIGF-1. The mutations of M195 and M577 did not affect viability and could not be complemented by the PMT1 or PMT2 genes. The mutant phenotype of strain M195 was reconstituted in an in vitro system, in which a hIGF-1-derived peptide encompassing residues 24-34 was not used as acceptor for mannosylation, while unrelated peptides were glycosylated at wild-type levels. hIGF-1 glycosylation was drastically reduced in pmt1 disruptants and to a lesser extent in pmt2 disruptants, suggesting interaction between the PMT gene products and components mutated in M195 and M577 cells. The results suggest that mutations may only affect O-glycosylation of a specific subset of secreted proteins in yeast.

Amino Acid Sequence↗
PubMed1991

The carboxyl terminus of Scg1, the G alpha subunit involved in yeast mating, is implicated in interactions with the pheromone receptors.

The carboxyl termini of alpha subunits of mammalian G proteins have been implicated in receptor interactions. We have used a genetic analysis to test such a role for the carboxyl terminus of Scg1, the alpha subunit involved in the yeast pheromone response pathway. A 22-amino-acid truncation (scg1Amb451) resulted in defects in growth and cellular morphology. This phenotype is similar to the null phenotype and represents constitutive activation of the pheromone response pathway; it could result from various effects, including protein instability or constitutive guanine nucleotide exchange, as reported for some altered mammalian G alpha s constructs. A 5-amino-acid truncation (SCG1Och468) resulted in pheromone response and mating defects in both a and alpha cells, which is consistent with defects in interactions with the pheromone receptors. Lysine-to-proline mutations near the carboxyl terminus (SCG1Pro467 and SCG1Pro468) resulted in pheromone response and mating defects, the severity of which differed in a and alpha cells. This differential effect in the two mating types suggests that the specificity for the interactions with the two pheromone receptors may involve different residues of the Scg1 carboxyl terminus. Mutations leading to constitutive activation of the pathway were recessive, whereas mutations that result in decreased pheromone response and mating were partially dominant. These relationships are consistent with the model for the mechanism of action of the G protein subunits in the pheromone response pathway and indicate the importance of the stoichiometry of components of this system.

Base Sequence↗
PubMed1991

Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner.

Cell Cycle↗
PubMed1994

Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities.

Biological Transport, Active↗
PubMed1997

The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor.

In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone. M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Afr1p regulates the Saccharomyces cerevisiae alpha-factor receptor by a mechanism that is distinct from receptor phosphorylation and endocytosis.

The alpha-factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor. AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p. To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis. AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation. The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors. These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis. Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for alpha-factor. Thus, Afr1p prevents alpha-factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways.

Cell Division↗
PubMed2000

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis.

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.

Cell Fractionation↗