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PubMed1991

Inhibition of glycogen synthesis in Saccharomyces cerevisiae by the mating pheromone alpha-factor.

Treatment of the yeast Saccharomyces cerevisiae with the pheromone alpha-factor caused an inhibition of glycogen synthesis in MATa haploid cells but not in MAT alpha cells or MATa/MAT alpha diploid cells. The concentration of alpha-factor required for a half-maximal inhibition was comparable to that required for the induction of the FUS1 gene. Strains containing a disruption in ste2 or ste12 or temperature-sensitive mutations in ste4, ste7, or ste11 continued to divide and to accumulate glycogen in the presence of alpha-factor. In contrast, inhibition of glycogen occurred upon exposure to mating pheromone of far1 mutants which, under this condition, fail to arrest in G1 and continue to divide while simultaneously undergoing the transcriptional induction and morphological changes typical of mating cells. The inhibition of glycogen accumulation by alpha-factor persisted in a strain lacking glycogen phosphorylase (EC 2.4.1.1), which ruled out the participation of this enzyme in the pheromone response. Glycogen synthase (EC 2.4.1.11) from a cells treated with alpha-factor was found primarily in the glucose 6-phosphate-dependent (inactive) form whereas the total activity was unaltered. This indicates that the action of mating pheromone is mainly to inhibit the interconversion of the inactive glucose 6-phosphate-dependent form to the active glucose 6-phosphate-independent form of glycogen synthase without affecting the concentration of the enzyme.

Amino Acid Sequence↗
PubMed1987

Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F' factor.

Filter matings between E. coli K-12 strains carrying an F'::Tn5,Tn9 factor with H. influenzae Rd strains gave rise to kanamycin-chloramphenicol-resistant H. influenzae strains at a frequency of approximately 10(-6). Transfer of the F' factor to H. influenzae was verified by expression of unselected markers in H. influenzae (lac+ or cotransfer of the nonselected antibiotic resistance), physical presence of a high-molecular-weight plasmid in recipient H. influenzae cells, and detection by Southern hybridization analysis of DNA sequences specific for the F' factor replication and partition functions in recipient H. influenzae cells. H. influenzae (F' Tn5,Tn9) strains were capable of transferring kanamycin and chloramphenicol resistances to other H. influenzae strains and were capable of mobilizing H. influenzae chromosomal markers at a low frequency. Insertion of a Tn5 element in the H. influenzae genome near the novobiocin resistance gene increased the frequency of transfer of novobiocin resistance about 30-fold. Transfer of other chromosomal markers also increased, although to a lesser extent, and ordered transfer of chromosomal markers could be demonstrated. Gene transfer was insensitive to DNase I, and transfer of chromosomal (but not F' factor) markers was dependent on the H. influenzae rec-1 and rec-2 gene functions.

Chromosome Mapping↗
PubMed1985

Secretion of mature mouse interleukin-2 by Saccharomyces cerevisiae: use of a general secretion vector containing promoter and leader sequences of the mating pheromone alpha-factor.

We have constructed a general expression vector which allows the synthesis and secretion of processed gene products in Saccharomyces cerevisiae. This vector contains yeast DNA, including the promoter of the mating pheromone (alpha-factor), its downstream leader sequence, and the TRP5 terminator. A cDNA [encoding mature mouse interleukin-2 (IL-2); Yokota et al., Proc. Natl. Acad. Sci. USA 82 (1984) 68-72] was fused immediately downstream to the alpha-factor leader sequence. The resulting recombinant plasmid directed the synthesis of mature mouse IL-2 in S. cerevisiae, with most of the T-cell growth-factor (TCGF) activity secreted into the culture fluid and extracellular space. TCGF activities in the cell extract, as well as in the culture fluid, increased in parallel with cell growth. Production of mature mouse IL-2 was inhibited by tunicamycin (TM), with precursor molecules accumulating in the cell extract. The precursor was processed accurately at the junction between the alpha-factor peptide leader sequence and the coding sequence downstream, yielding mature IL-2. The Mr of the secreted mouse IL-2 determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was 17 kDal, a value expected for the mature mouse IL-2 polypeptide based on the nucleotide (nt) sequence.

Amino Acid Sequence↗
PubMed1985

Sexual Agglutination in the Unicellular Green Alga Chlamydomonas eugametos: Identification and Properties of the Mating Type plus Agglutination Factor.

Gametes of the unicellular green alga Chlamydomonas eugametos agglutinate via their flagella. The mating type plus agglutination factor was solubilized by relatively mild treatments such as a short pH shock or an osmotic shock indicating that it is an extrinsic membrane component. It was also extracted in the nonionic detergent Triton X-100. A simple two-step procedure consisting of gel filtration over Sepharose 4B-cross-linked followed by anion exchange chromatography of the void volume yielded an electrophoretically pure preparation of a single high molecular weight glycoprotein. The agglutination factor sedimented as a 9.3 S particle (assuming a density of 1.50) in sucrose gradients. This low value, compared with the high apparent molecular weight seen during gel filtration and electrophoresis, suggests that the agglutination factor is a rod-like molecule. This was confirmed by viewing rotary-shadowed preparations in the electron microscope. A population of long slender molecules was revealed (328 +/- 20 nanometers), many of which had a knob at one end and a flexible region about one fourth of the length from the other end.

Journal Article↗
PubMed1991

Inhibition of membrane Ca2(+)-ATPase of Saccharomyces cerevisiae by mating pheromone alpha-factor in vitro.

Plasma membrane Ca2(+)-ATPase of Saccharomyces cerevisiae was solubilized and partially purified by calmodulin-affinity chromatography. The activity of Ca2(+)-ATPase isolated from MATa cells was inhibited by physiological concentrations of the mating pheromone alpha-factor in a dose-dependent manner. The enzyme prepared from a receptor-deficient sterile mutant cells (delta ste-2) was similarly inhibited by alpha-factor, but the enzyme from MAT alpha cells was resistant to the mating pheromone. We suggest that the inhibition may be involved in the alpha-factor-induced increase of Ca2+ uptake reaction of MATa cells.

Bacterial Proteins↗
PubMed1992

Mating type switching in the tetrapolar basidiomycete Agrocybe aegerita.

The study of fruiting in the basidiomycete Agrocybe aegerita has shown that some haploid homokaryotic strains can spontaneously switch their mating specificities at the two unlinked A and B mating type factors. This event causes the dikaryotisation of primary homokaryons without plasmogamy and leads to the differentiation of sporulating fruit-bodies (pseudo-homokaryotic fruiting). For each mating type factor, the genetic analyses have revealed that: (1) parental and switched mating types segregate meiotically as Mendelian markers, (2) a total of six switched mating type factors (two parental and four nonparental) were obtained from a wild strain, (3) most of the nonparental factors have specificities differing from those of a large series of wild factors, (4) strains with the same expressed mating type can generate different specificities, (5) switching is always restricted to the same mating type in a homokaryon, (6) nonparental types can switch again, and (7) meiosis fixes the specificities to which switching can occur. This suggests, for the first time in filamentous fungi, the existence of a mechanism analogous to the mating type switching in yeasts. We hypothese that both A and B mating type regions in A. aegerita are constituted of three loci, one specialized in expression and two other carrying silent information. Mating type switching in homokaryotic strains would occur by copy transposition of silent A and B information into the expression loci. Moreover, we propose that during meiosis the silent loci are substituted by copies of the expressed loci.

Basidiomycota↗
PubMed2002

Mutagenic mapping of helical structures in the transmembrane segments of the yeast alpha-factor receptor.

The alpha-mating pheromone receptor encoded by the yeast STE2 gene is a G protein coupled receptor that initiates signaling via a MAP kinase pathway that prepares haploid cells for mating. To establish the range of allowed amino acid substitutions within transmembrane segments of this receptor, we conducted extensive random mutagenesis of receptors followed by screening for receptor function. A total of 157 amino acid positions in seven different mutagenic libraries corresponding to the seven predicted transmembrane segments were analyzed, yielding 390 alleles that retain at least 60 % of normal signaling function. These alleles contained a total of 576 unique amino acid substitutions, including 61 % of all the possible amino acid changes that can arise from single base substitutions. The receptor exhibits a surprising tolerance for amino acid substitutions. Every amino acid in the mutagenized regions of the transmembrane regions could be substituted by at least one other residue. Polar amino acids were tolerated in functional receptors at 115 different positions (73 % of the total). Hydrophobic amino acids were tolerated in functional receptors at all mutagenized positions. Substitutions introducing proline residues were recovered at 53 % of all positions where they could be brought about by single base changes. Residues with charged side-chains could also be tolerated at 53 % of all positions where they were accessible through single base changes. The spectrum of allowed amino acid substitutions was characterized in terms of the hydrophobicity, radius of gyration, and charge of the allowed substitutions and mapped onto alpha-helical structures. By comparing the patterns of allowed substitutions with the recently determined structure of rhodopsin, structural features indicative of helix-helix interactions can be discerned in spite of the extreme sequence divergence between these two proteins.

Alleles↗
PubMed1999

Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides.

The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094].

Algorithms↗
PubMed1982

Yeast mating pheromone activates mammalian gonadotrophs: evolutionary conservation of a reproductive hormone?

alpha-Factor, a tridecapeptide mating pheromone of yeast (Saccharomyces cerevisiae), has extensive sequence homology with the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH). Both synthetic and natural preparations of alpha-mating factor were found to bind specifically to rat pituitary GnRH receptors and to stimulate the release of luteinizing hormone from cultured gonadotrophs. The ability of the yeast pheromone to reproduce the biological actions of GnRH in the mammalian pituitary gland indicates that the structural and functional properties of GnRH-related peptides may have been highly conserved during evolution.

Animals↗
PubMed1991

In-vitro processing of yeast alpha-factor leader fusion proteins using a soluble yscF (Kex2) variant.

The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the alpha-factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscFs). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for alpha-factor leader-hIGF1 and alpha-factor leader-hirudin fusion proteins.

Colorimetry↗
PubMed1987

Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing.

The yeast KEX1 gene product has homology to yeast carboxypeptidase Y. A mutant replacing serine at the putative active site of the KEX1 protein abolished activity in vivo. A probable site of processing by the KEX1 product is the C-terminus of the alpha-subunit of killer toxin, where toxin is followed in the precursor by 2 basic residues. Processing involves endoproteolysis following these basic residues and trimming of their C-terminal by a carboxypeptidase. Consistent with the KEX1 product being this carboxypeptidase is its role in alpha-factor pheromone production. In wild-type yeast, KEX1 is not essential for alpha-factor production, as the final pheromone repeat needs no C-terminal processing. However, in a mutant in which alpha-factor production requires a carboxypeptidase, pheromone production is KEX1-dependent.

Amino Acid Sequence↗
PubMed1994

AFC1, a LAMMER kinase from Arabidopsis thaliana, activates STE12-dependent processes in yeast.

In the yeast Saccharomyces cerevisiae a kinase cascade activates the transcription factor STE12 leading to mating in haploid cells and pseudohyphal growth in diploid cells. To investigate related signal transduction pathways in higher plants, we have isolated a putative protein kinase gene from Arabidopsis thaliana that restores STE12-dependent functions to yeast with mutations in this signal transduction pathway. This Arabidopsis gene, AFC1, induces three STE12-dependent processes even in signal transduction-defective yeast strains: mating-specific gene expression in haploid yeast, mating of haploid yeast to yield diploids, and pseudohyphal growth in diploid yeast. AFC1 has no effect on transcription of the STE12 gene and, instead, is likely to activate the STE12 protein. However, AFC1 has only limited homology to FUS3 and KSS1, the endogenous yeast kinase regulators of STE12. AFC1 is a member of a recently described CDC2-related kinase subfamily, the LAMMER kinases. A close AFC1 homolog, AFC2, lacks STE12 activation phenotypes, indicating the specificity of AFC1. The phenotypes of AFC1 in yeast provide us with tools to elucidate the role of this kinase in Arabidopsis.

Amino Acid Sequence↗
PubMed1997

KEX2 influences Candida albicans proteinase secretion and hyphal formation.

Candida albicans possesses at least seven differentially expressed genes that encode virulence-related secretory aspartyl proteinases (Saps). Sap DNA sequences predict post-translational processing at lysine-arginine residues in the preproteins, reminiscent of the maturation of Saccharomyces cerevisiae alpha-factor, where a prepropolypeptide is converted into a biologically active pheromone by Kex2, a subtilisin-like proprotein convertase. To investigate involvement of a C. albicans KEX2 homologue in Sap activation, a genetic selection was performed based on KEX2 function. A kex2 strain of S. cerevisiae was transformed with a C. albicans genomic DNA library and screened for the production of active alpha-factor. Positive clones were assayed for killer toxin activity, another Kex2-dependent phenotype. Plasmids that rescued both defects contained a sequence encoding a protein homologous to S. cerevisiae Kex2. Both alleles of the C. albicans KEX2 were inactivated by successive mutations. Null mutants continued to secrete active Sap2; however, the enzyme was abnormally processed and secreted at reduced levels. Unexpectedly, null mutants were incapable of forming hyphae, instead differentiating into aberrantly shaped cells. The ability to normally process Sap2 and form hyphae was restored upon transformation of null mutants with a KEX2-containing plasmid.

Amino Acid Sequence↗
PubMed1986

Improved secretion of heterologous proteins by Saccharomyces cerevisiae: effects of promoter substitution in alpha-factor fusions.

The effects of promoter strength on secretion of a heterologous protein, somatomedin-C (SMC), by the yeast Saccharomyces cerevisiae were studied by using the promoters of the MF alpha 1, ACT, and CYC1 genes to control expression of alpha-factor/SMC gene fusions. When a low-copy centromere vector was used to carry the gene fusions in yeast transformants, the greatest secretion was obtained with the MF alpha 1 promoter construction and the least with the CYC1 promoter construction. Unexpectedly, using two types of multicopy vectors, the greatest secretion was obtained with the CYC1 promoter construction and the least with the MF alpha 1 promoter construction. The decrease in secretion by the strongest promoter construction (MF alpha 1 promoter) on multicopy vectors was associated with a decrease in SMC mRNA during growth, a decrease in vector copy number, a decrease in vector stability, and a decrease in transformation frequency. The results demonstrate that, unlike in intracellular expression, promoter strength is not simply related to secretion expression levels. Selection against oversecreting cells during growth may explain the reduced secretion efficiency of strong promoter constructions.

Actins↗
PubMed1998

A yeast t-SNARE involved in endocytosis.

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.

Amino Acid Sequence↗