Secretion of heterologous proteins directed by the yeast alpha-factor leader.
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Amino Acid Sequence↗
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Results for “Mating Factor”
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Mating in the heterothallic haploid yeast Clavispora opuntiae, with special reference to mating type imbalances in local populations.
Mating was studied in the haploid, heterothallic yeast Clavispora opuntiae to assess the importance of nutritional, genetic, and other factors that may favour mating and recombination. Local populations of this yeast generally exhibit dramatic inequalities in mating type distributions, suggesting that mating is rare in nature even though most isolates mate freely in the laboratory. The absence of assimilable nitrogen is prerequisite to mating competence, presumably by causing G1 arrest. Maximum mating competence is found in cells entering stationary phase in nitrogen-limited media. Unlike the vast majority of mating yeasts, C. opuntiae does not appear to produce diffusible mating factors (sex pheromones), and mating-competent cells do not undergo sexual agglutination. Pairwise cell contact appears to be the only signal that triggers the sexual process in this case. In order to determine if mating type imbalances in nature are caused by reduced fertility of 'consanguine' crosses, meiotic recombination was measured in pairs of strains that varied in their genetic distances as indicated by restriction mapping. That hypothesis was rejected, as recombination efficiency decreased with increasing genetic distance. We conclude that the rarity of mating in local populations is exacerbated by the stringent physical (pairwise cell contact) and nutritional (nitrogen depletion) conditions that will allow mating to proceed. Parallels are drawn with mating patterns observed in Clavispora lusitaniae.
Crosses, Genetic↗
Autocrine mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi.
Diffusible polypeptide pheromones (formerly referred to as mating-type factors, sex factors or gamones), which distinguish otherwise morphologically identical vegetative cell (mating) types from one another, are produced by some species of ciliates. Their most striking effect can be observed by exposing cells of one type to a pheromone secreted by another co-specific cell type. In the presence of this 'non-self' signal, these cells interrupt their vegetative life to unite temporarily in mating pairs. Thus ciliate pheromones have traditionally been associated only with mating induction. However, the identification of autocrine pheromone receptors suggests a broader role, which is supported by the hypothesis that ciliates evolved their mating-type mechanism for pursuing self-recognition. We now report studies, in the cosmopolitan marine sand-dwelling protozoan ciliate Euplotes raikovi, demonstrating that these molecules promote the vegetative reproduction (mitogenic proliferation or growth) of the same cells from which they originate. As, understandably, such autocrine pheromone activity is primary to that of targeting and inducing a foreign cell to mate (paracrine functions), this finding provides an example of how the original function of a molecule can be obscured during evolution by the acquisition of a new one.
Amino Acid Sequence↗
Properties of F' factor deoxyribonucleic acid transferred from ultraviolet-irradiated donors: photoreactivation in the recipient and the influence of recA, recB, recC, and uvr genes.
Ultraviolet (UV)-irradiated Escherichia coli K-12 F lac(+) donors transfer damaged F' factors when mated with female cells. Exposure of the zygotes to white light after mating can cause the photoreactivation of the damaged transferred F' factors. In so far as the photoreactivation is specific for pyrimidine dimers, these experiments indicate the presence of UV-induced dimers in the transferred F' factor deoxyribonucleic acid (DNA). These damaged F' factors are remarkably stable in recA recipients, having a half-life for susceptibility to photoreactivation of 1.5 to 2 hr. This is judged by the formation of Lac(+) colonies, all of which are male secondary donors. In crosses with wild type and uvrA, recB, or recC mutant recipients, however, the Lac(+) colonies are predominantly recombinants and photoreactivation is not detected. The extent of DNA synthesis resulting from transfer of F' episomes from irradiated donors suggests that a complementary strand is formed on the damaged template. Photoreactivation behavior and sedimentation properties are used to deduce properties of such damaged episomes. We conclude that the complementary strand is discontinuous directly opposite dimers in the transferred strand. This structure may be an intermediate in the recombinational event sequence in Rec(+) recipients.
Conjugation, Genetic↗
Assortative mating in a Spanish population: effects of social factors and cohabitation time.
Assortative mating for 22 anthropometric and body composition characteristics and social indicators was studied in a Spanish sample of 114 married couples. Significant spousal similarity was found for occupation, educational level and number of siblings. Sex-age adjusted spouse correlations were significant for stature, ileospinal height, total arm length, and biacromial breadth. Spouse correlations were not altered after allowance for socioeconomic effects. When couples were grouped according to marriage duration, differences in mate correlations for fatness were detected, suggesting a cohabitational effect on spouse resemblance.
Adult↗
The alpha-factor receptor C-terminus is important for mating projection formation and orientation in Saccharomyces cerevisiae.
Successful mating of MATa Saccharomyces cerevisiae cells is dependent on Ste2p, the alpha-factor receptor. Besides receiving the pheromone signal and transducing it through the G-protein coupled MAP kinase pathway, Ste2p is active in the establishment and orientation of the mating projection. We investigated the role of the carboxyl terminus of the receptor in mating projection formation and orientation using a spatial gradient assay. Cells carrying the ste2-T326 mutation, truncating 105 of the 135 amino acids in the receptor tail including a motif necessary for its ligand-mediated internalization, display slow onset of projection formation, abnormal shmoo morphology, and reduced ability to orient the mating projection toward a pheromone source. This reduction was due to the increased loss of mating projection orientation in a pheromone gradient. Cells with a mutated endocytosis motif were defective in reorientation in a pheromone gradient. ste2-Delta296 cells, which carry a complete truncation of the Ste2p tail, exhibit a severe defect in projection formation, and those projections that do form are unable to orient in a pheromone gradient. These results suggest a complex role for the Ste2p carboxy-terminal tail in the formation, orientation, and directional adjustment of the mating projection, and that endocytosis of the receptor is important for this process. In addition, mutations in RSR1/BUD1 and SPA2, genes necessary for budding polarity, exhibited little or no defect in formation or orientation of mating projections. We conclude that mating projection orientation depends upon the carboxyl terminus of the pheromone receptor and not the directional machinery used in budding.
Cell Differentiation↗
Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae. An alpha-factor inducible gene.
A method has been developed for the rapid purification of yeast calmodulin in high yield. Using a 125I-labeled calmodulin SDS/PAGE gel overlay procedure with either yeast or bovine calmodulin, we show that the bovine and yeast proteins recognize the same proteins in total yeast extracts. However, yeast calmodulin does not bind to many of the proteins in vertebrate cells identified using bovine calmodulin. A lambda gt11 yeast genomic expression library was screened with yeast or bovine brain 125I-calmodulin to identify sequences derived from calmodulin binding proteins. Twelve clones were recovered, all containing a common DNA insert; all bound calmodulin in a Ca(2+)-dependent manner. The complete coding sequence was recovered and sequenced. The predicted protein sequence show greater than 50% identity to the A subunit of vertebrate protein phosphatase 2B. The gene was designated CMP1 and shown to reside on chromosome IV. Disruption or over-expression of CMP1 have no obvious phenotype; yeast appears to contain one or more CMP1-related genes. The protein product of the CMP1 gene is elevated by alpha-factor treatment, suggesting an involvement of protein phosphatase 2B in the mating response.
Amino Acid Sequence↗
Structure-activity relationships of the yeast alpha-factor.
The yeast Saccharomyces cerevisiae produces a peptide pheromone, termed the alpha-factor, as a prelude to sexual conjugation. Haploid MAT alpha-cells, but not haploid MAT a-cells or MAT a/alpha-diploids, produce this tridecapeptide of the structure: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr. Structural analogues of the alpha-factor have been prepared with alterations in many of the residues, derivatized peptides have been synthesized, and truncated and elongated peptides have been studied. These peptides have been analyzed for their biological activities by various assays. Mutants of S. cerevisiae have been isolated that do not respond to alpha-factor or are supersensitive to the pheromone and its analogues. The mating system of S. cerevisiae provides a powerful model in which genetics, biochemistry, and molecular biology can be used to unravel the mysteries of peptide hormone structure and function.
Amino Acid Sequence↗
The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis.
In Ustilago maydis, the a and b mating type loci regulate cell fusion, filamentous growth and pathogenicity. The a locus encodes a pheromone-based cell recognition system, and the b locus specifies two homeodomain proteins. The expression of all genes in the a and b loci is induced by pheromone. We have identified a HMG protein (Prf1) that binds sequence specifically to pheromone response elements present in the a and b loci. prf1 mutants do not express the a and b genes and are sterile. The disruption of prf1 in pathogenic haploid strains results in a loss of pathogenicity. The constitutive expression of the b genes restores pathogenicity and induces filamentous growth in the absence of the pheromone signal. These results provide evidence that pheromone signalling, filamentous growth and pathogenic development are linked through Prf1.
Amino Acid Sequence↗
Yeast peptide pheromones, a-factor and alpha-factor, activate a common response mechanism in their target cells.
We show that in yeast the cell type specificity of pheromone response is determined solely by the species of receptor that a cell synthesizes. The two receptor-pheromone interactions are functionally interchangeable and involve the creation of a common intracellular signal. In particular, we find that provision of a-factor receptor or alpha-factor receptor in mat alpha 1 mutants, which normally do not express either receptor or any other a- or alpha-specific products, allows response to the appropriate pheromone. Moreover, provision of a-factor receptor in a cells lacking alpha-factor receptor restores mating competence to those cells. Finally, an aspect of pheromone response that is normally unique to a-factor action on alpha cells--increased transcription from the alpha-specific STE3 gene--can also be observed following alpha-factor treatment of pseudo-a cells (mat alpha 2 ste3 ste13), special mutants that respond to alpha-factor and also have an active STE3 promoter.
Genes, Fungal↗
Properties of Barrier, a novel Saccharomyces cerevisiae acid protease.
We have studied the specificity of Barrier, a protease secreted by Saccharomyces cerevisiae, towards its natural substrate alpha-factor, a tridecapeptide mating pheromone. Sub-fragments of alpha-factor synthesized or prepared by cyanogen bromide cleavage and a related pheromone from Saccharomyces kluveri were studied as potential substrates or competitive inhibitors. None of the tested peptides was a potent inhibitor or substrate for Barrier. Barrier shares extensive sequence similarity to the active site residues of aspartyl proteases but universal irreversible inhibitors of this class of enzymes failed to inactivate Barrier, suggesting that it is a novel fungal aspartyl protease.
Amino Acid Sequence↗
Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest.
Mating response between opposite mating types of Saccharomyces cerevisiae is dependent upon alpha factor, a tridecapeptide, and a-factor, an isoprenylated, methyl esterified dodecapeptide whose interaction with the alpha target cell has not been characterized. We report on the first biochemical and physiological evidence of an alpha-mating-type-specific a-factor-degrading activity. Radioiodinated a-factor was used to identify the a-factor-degrading activity, which is cell associated, endoproteolytic, and not required for response to pheromone. a-factor degradation was not energy dependent, nor did it require pheromone internalization or interaction with its receptor. Phenylmethylsulfonyl fluoride and tosyl-L-arginyl-methyl ester inhibited degradation of a-factor and increased the time required by alpha cells to recover from a-factor-induced growth arrest and morphological alteration, providing evidence that a-factor degradation plays a role in the recovery of alpha cells from the pheromone response.
Amino Acid Sequence↗
Yeast surface display for directed evolution of protein expression, affinity, and stability.
The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones. It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed. With library plasmid DNA in hand, the time to plated candidate yeast display mutants is typically 2-3 weeks. Each of the experimental approaches required for this method is fairly standard: yeast culture, immunofluorescent labeling, flow cytometry. Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance. However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method. Yeast display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to yeast two-hybrid or phage display methodologies.
Amino Acid Sequence↗
A MATERNAL FACTOR WHICH BREAKS PATERNAL CHROMOSOMES.
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Animals↗
Dynamic localization of protein phosphatase type 1 in the mitotic cell cycle of Saccharomyces cerevisiae.
Protein phosphatase type I (PP1), encoded by the single essential gene GLC7 in Saccharomyces cerevisiae, functions in diverse cellular processes. To identify in vivo subcellular location(s) where these processes take place, we used a functional green fluorescent protein (GFP)-Glc7p fusion protein. Time-lapse fluorescence microscopy revealed GFP-Glc7p localizes predominantly in the nucleus throughout the mitotic cell cycle, with the highest concentrations in the nucleolus. GFP-Glc7p was also observed in a ring at the bud neck, which was dependent upon functional septins. Supporting a role for Glc7p in bud site selection, a glc7-129 mutant displayed a random budding pattern. In alpha-factor treated cells, GFP-Glc7p was located at the base of mating projections, again in a septin-dependent manner. At the start of anaphase, GFP-Glc7p accumulated at the spindle pole bodies and remained there until cytokinesis. After anaphase, GFP-Glc7p became concentrated in a ring that colocalized with the actomyosin ring. A GFP-Glc7-129 fusion was defective in localizing to the bud neck and SPBs. Together, these results identify sites of Glc7p function and suggest Glc7p activity is regulated through dynamic changes in its location.
Actomyosin↗
Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae.
Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.
Actins↗
Factors influencing age at first mating in purebred Swedish Landrace and Swedish Yorkshire gilts.
The aim of the present study was to retrospectively analyze causes of the variation in age at first mating in Swedish Landrace (L) and Swedish Yorkshire (Y) gilts. Production traits including growth rate from birth to 100kg body weight and backfat thickness at 100kg body weight were also studied. Data analyzed were obtained from 11 L and 11 Y nucleus herds and included gilts born during a 5-year-period from October 1993 until September 1998. The complete data set included information on 14,761 gilts (6997 L and 7764 Y). Traits analyzed included age of gilt at first mating, growth rate and backfat thickness. Seven statistical models were used for analyzing the data. Factors included were gilt breed, birth month, parity number and size of the litter in which the gilt was born as well as their interactions. Compared with Y gilts, L gilts grew faster (571 versus 556 g/day; P<0.001), had a thinner backfat (11.9 versus 12. 3mm; P<0.001) at 100kg body weight and were 12 days younger at first mating (237 versus 249 days; P<0.001). Birth month significantly (P<0.001) influenced age at first mating, growth rate and backfat thickness. Gilts born from smaller litters were mated at younger age than gilts born from larger litters even when age at first mating was adjusted for the effect of growth rate and backfat thickness. Growth rate of the gilts decreased when 'birth litter size' increased. Gilts born from primiparous sows grew slower, had a thinner backfat at 100kg body weight and were older at first mating compared with gilts born from multiparous sows. Gilts with a higher growth rate were younger at first mating than those with a lower growth rate. Gilts with a thicker backfat at 100kg body weight were mated earlier than the thin ones. However, the effect of growth rate on age at first mating was more pronounced in the gilts with a thinner backfat rather than the ones with a thicker backfat.
Age Factors↗
Management of atopic dermatitis in children; control of maternal rejection factor.
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Child↗