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J Thorner

Publications and source records attributed to J Thorner.

At least 109 records · Page 6Linked to original sources
PubMed1984

Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway.

Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway. In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro. At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates. In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed. The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus. Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5).

Genotype↗
PubMed1984

Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor.

S. cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor. Both molecules are excised from larger precursor polypeptides. In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly. In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form. All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues. Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses. The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation. The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo. Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase.

Arginine↗
PubMed1984

Molecular cloning of hormone-responsive genes from the yeast Saccharomyces cerevisiae.

A method for identifying yeast genes whose transcription is differentially regulated was developed. The technique is based on incorporation of the analog 4-thiouridine into nascent RNAs, which allows their purification. The purified RNAs are used to prepare cDNA copies for screening of genomic DNA libraries by hybridization. Using this procedure, several cloned yeast DNA segments were found whose transcription in MATa haploids in vivo is apparently modulated in dramatic fashion within 10-15 min after exposure to the mating pheromone, alpha factor. Subsequent analysis indicated that these sequences fall into three major classes: (i) genes expressed in vegetatively growing cells that are no longer transcribed after alpha-factor administration ("turn-off" genes); (ii) genes whose expression is increased 10- to 20-fold after exposure of the MATa cells to alpha factor ("turn-up" genes); and (iii) genes that are expressed only after alpha-factor treatment ("turn-on" genes). The first class may encode products required for cell cycle progression; the third class may code for products uniquely involved in the mating process.

Animals↗
PubMed1983

Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase.

Alpha factor mating pheromone is a peptide of 13 amino acids secreted by Saccharomyces cerevisiae alpha cells. Nonmating ("sterile," or ste) alpha-cell mutants bearing defects in the STE13 gene do not produce normal alpha factor, but release a collection of incompletely processed forms (alpha factor) that have a markedly reduced specific biological activity. The major alpha-factor peptides have the structures H2N-GluAlaGluAla-alpha factor and H2N-AspAlaGluAla-alpha factor. The ste13 mutants lack a membrane-bound heat-stable dipeptidyl aminopeptidase (DPAPase A) that specifically cleaves on the carboxyl side of repeating -X-Ala- sequences. Absence of DPAPase A and the other phenotypes of a ste13 lesion cosegregate in genetic crosses. The cloned STE13 gene on a plasmid causes yeast cells to overproduce DPAPase A severalfold. A different cloned DNA segment, which weakly suppresses the ste13 defects, causes overproduction of a heat-labile activity (DPAPase B) by about tenfold. Other experiments indicate that DPAPase A action may be rate-limiting for alpha-factor maturation in normal alpha cells.

Amino Acids↗
PubMed1983

An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast.

The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-alpha-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.

Amino Acid Sequence↗
PubMed1983

Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae.

MAT alpha haploids with mutations in the STE13 or KEX2 gene, and MATa haploids with mutations in the STE6 or STE14 gene, do not mate with wild-type cells of the opposite mating type. We found that such mutants were able to mate with partners that carry mutations (sst1 and sst2) that cause cells to be supersensitive to yeast mating pheromone action. Mating ability of MAT alpha ste13 and MAT alpha kex2 mutants could also be restored by adding normal MAT alpha cells to mating mixtures or by adding just the appropriate purified pheromone (alpha-factor). Therefore, the mating deficiencies caused by the ste13 and kex2 lesions, and by inference, the ste6 and ste14 mutations, appear to result only from secretion of an insufficient amount of pheromone or a nonfunctional pheromone.

Animals↗
PubMed1983

A functional prepro-alpha-factor gene in Saccharomyces yeasts can contain three, four, or five repeats of the mature pheromone sequence.

The chromosomal region containing a structural gene for the mating pheromone precursor prepro-alpha-factor was examined in a variety of Saccharomyces yeasts by using a cloned putative prepro-alpha-factor gene of Saccharomyces cerevisiae as the probe. Analysis by restriction endonuclease digestion and Southern blot hybridization indicated that the physical arrangement of this region is highly conserved in all the Saccharomyces species analyzed, but displays length polymorphisms of limited size (50 to 60 base pairs). The observed polymorphisms were shown to be due solely to differences in the number of tandemly arranged spacer peptide/pheromone units within the coding sequence of these genes. Analysis of polyadenylated RNA indicated that these genes specified RNA transcripts and that these RNA molecules could be translated in vitro into prepro-alpha-factor polypeptides immunoprecipitable with anti-alpha-factor antibodies. The sizes of both the mRNAs and the proteins synthesized from them reflected exactly the differences observed in the lengths of the genes. These findings demonstrate conclusively that the putative prepro-alpha-factor DNA cloned from S. cerevisiae, as well as the sequences detected in the other Saccharomyces species, are indeed expressed and functional genes, and suggest that proper proteolytic processing of prepro-alpha-factor is unaffected by the number of pheromone repeats encoded within this precursor protein.

Animals↗
PubMed1983

Cell interactions and regulation of cell type in the yeast Saccharomyces cerevisiae.

Examination of the control of cell type in yeast at the molecular level and understanding of the biochemical basis of the cell-cell interactions involved in the mating process are clearly entering an extremely productive and exciting period. The tools and opportunities are now available to answer fundamental questions with regard to the mechanism of differential gene expression in eukaryotic cells by using cloned a-specific, alpha-specific, and haploid-specific genes as the probes. Basic questions concerning eukaryotic chromosome structure and organization can be addressed by elucidating the properties of the SIR/MAR regulators and their mode of action. Furthermore, the availability both of cloned MAT, HML, and HMR regions and of the HO gene will provide the material for unravelling the enzymology of the DNA transposition that occurs during mating type interconversion. The isolation of the structural genes for the pheromones and mutations that block pheromone production will provide useful information on how extracellular hormones are synthesized, processed, and secreted by eukaryotic cells. Moreover, the apparent mode of action of the phermonones through cyclic AMP as an intracellular "second messenger," and the genetic and biochemical tractability of yeast cells, may allow tracing of the entire pathway of hormonal regulation of a eukaryotic cell division cycle. These and other studies of the developmental biology of yeast cells will provide more important insights into fundamental aspects of the genetic control of developmental processes in eukaryotic cells.

Animals↗
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↗
PubMed1982

Effect of halogenated pyrimidine 5'-mononucleotides on dTMP-permeable yeast strains and the isolation and characterization of resistant mutants.

Unlike wild-type Saccharomyces cerevisiae, yeast cells carrying the tup 7 mutation are able to take up exogenously-supplied dTMP. The tup 7 mutant was also found to be dramatically sensitive to growth inhibition by FdUMP and BrdUMP. The exclusive mode of action of FdUMP in such strains was shown to be inhibition of thymidylate synthetase. Spontaneously-arising derivatives resistant to FdUMP and BrdUMP were isolated from the tup7 strain. Genetically, these mutations were recessive and defined three complementation groups (fdr1, fdr2, and bdr2), unlinked to the tup7 locus and to each other. No resistance mutations were obtained which mapped at the structural gene for thymidylate synthetase. Biochemical analysis of cells carrying these mutations showed that in the case of fdr2 and bdr2, in addition to an inability to transport dTMP, acid and alkaline phosphatase levels were affected, indicating that phosphatase expression and 5'-mononucleotide permeability are coordinately controlled. In contrast, the fdr1 mutation and a previously identified suppressor of dTMP-permeability, sot1, affected only 5'-mononucleotide uptake and may define components of the permease responsible for dTMP entry.

Drug Resistance↗
PubMed1982

Mutations in the pho80 gene confer permeability to 5'-mononucleotides in Saccharomyces cerevisiae.

Yeast mutants permeable to dTMP (tup) were selected and two new complementation groups (tup5 and tup7) were identified. Assay of the levels of both acid and alkaline phosphatase in cells grown under either repressing (5 mM PO4(-3) or derepressing (0.03 mM PO4(-3) conditions indicated that, in general, tup mutations cause cells to be defective in their regulation of phosphatase synthesis. In addition, three of the tup mutations (tup1, tup4 and tup7) displayed markedly elevated rates of inorganic phosphate transport. The tup7 locus was found to be tightly centromere-linked on the right arm of chromosome XV, and was shown to be allelic with the pho80 regulatory locus on the basis of both genetic and biochemical criteria. Analysis of other mutations known to affect phosphatase levels (pho) indicated that some also conferred permeability to dTMP. Possible allelic relationships between tup genes and certain of these pho mutations are discussed. Regardless of the culture conditions, wild-type strains were not permeable to dTMP; in contrast, it was found in the course of this work that normal yeast cells were permeable to dUMP and that dUMP permeability was regulated by the concentration of inorganic phosphate present in the medium used to grow the cells. Thus, permeability to 5'-mononucleotides appears to be under coordinate control with phosphatase synthesis.

Acid Phosphatase↗
PubMed1982

Exogenous dTMP utilization by a novel tup mutant of Saccharomyces cerevisiae.

The rate and extent of entry of dTMP were measured in strains of Saccharomyces cerevisiae carrying two new tup mutations (tup5 and tup7) and most of the other tup mutations which have been reported previously by others. The tup7 mutation allowed dramatically greater accumulation of dTMP than any of the other mutations tested. Specific labeling of DNA by [CH3-3H]dTMP, fate of the dTMP pool inside of the cells, and degradation of the dTMP in the culture medium were investigated in strains carrying the tup7 mutation. The extracellular dTMP was not appreciably degraded, and that accumulated intracellularly was readily phosphorylated to dTDP and dTTP. Under optimum labeling conditions, 60 to 80% of the total thymidylate residues in newly synthesized DNA were derived from the exogenously provided dTMP, even in the absence of a block in de novo dTMP biosynthesis. An apparent Km for entry of 2 mM dTMP was found. The tup7 mutation increased permeability to dTMP (and some other 5'-mononucleotides), but did not affect uptake of nucleosides and purine and pyrimidine bases. Uptake of dTMP could be almost completely inhibited by moderate concentrations of Pi. These findings and other observations suggest that entry of dTMP in strains carrying the tup7 mutation is mediated by a permease whose function in normal cells is the transport of Pi.

Biological Transport↗
PubMed1981

Thymidylate synthetase from Saccharomyces cerevisiae. Purification and enzymic properties.

Thymidylate synthetase of Saccharomyces cerevisiae was purified over 20,000-fold to apparent homogeneity by a procedure involving two new affinity methods and several precautions for avoiding proteolysis. Molecular weight of the native enzyme was about 65,000, as determined by gel filtration and velocity sedimentation. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight 30,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The purified enzyme exhibited normal Michaelis-Menten kinetics toward both substrates, with apparent Km values for dUMP and for (--)-5,10-methylene-tetrahydropteroylglutamate of 5 microM and 70 microM, respectively. When the pentaglutamyl form of the cofactor was used, its apparent Km was lower (7 microM), but Vmax was unaltered. Reaction kinetics and product inhibition studies were most consistent with an ordered mechanism, wherein dUMP is the first substrate to bind and 7,8-dihydropteroylglutamate is the first product released. Halogenated analogs of the nucleotide substrate were competitive inhibitors of the yeast enzyme, with apparent Ki values for 5-fluoro-dUMP of 5 nM and for 5-Br-dUMP of 10 microM. Analogs of the cofactor were also competitive inhibitors, with apparent Ki values for both methotrexate and aminopterin of about 20 microM. Cibacron blue, a dye used as the ligand in an affinity adsorbent for one of the purification steps, was a potent competitive inhibitor with respect to either substrate, yielding apparent Ki values of 4 nM for the nucleotide binding site and 40 nM for the cofactor binding site.

Hydrogen-Ion Concentration↗
PubMed1981

Adenosine 3',5'-phosphate phosphodiesterase and pheromone response in the yeast Saccharomyces cerevisiae.

Theophylline, aminophylline, and isobutylmethylxanthine, compounds reported to be inhibitors of adenosine 3',5'-phosphate (cAMP) phosphodiesterase, prevented the alpha-factor-induced cell cycle arrest of Saccharomyces cerevisiae a cells. To determine whether the in vivo effect of these methylxanthines on yeast pheromone response was related to their known biochemical mode of action, two assays for cAMP phosphodiesterase based on affinity of the product of the reaction (5'-AMP) for boronate groups were developed and were used to monitor the activity of the low Km cAMP phosphodiesterase present in yeast extracts. It was found that the relative efficacy of the methylxanthines as inhibitors of this enzyme in vitro was correlated with the degree to which they antagonized alpha-factor action in vivo. These results were consistent with our previous proposal that pheromone action involves a lowering of cAMP level in the target cell.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Yeast mating pheromone alpha factor inhibits adenylate cyclase.

The pheromone alpha factor, secreted by Saccharomyces cerevisiae cells of the alpha mating type, serves to synchronize the opposite mating type (a cells) at G1 as a prelude to fusion of the two cell types. We found that, in vitro, alpha factor inhibited the membrane-bound adenylate cyclase of these cells in a dose-dependent manner. Moreover, one class (ste5) of a cell mutants that grow normally at either 23 degrees or 34 degrees C but that are unable to respond to alpha factor or to mate at the higher temperature possessed an adenylate cyclase activity that was not inhibited by alpha factor at 34 degrees C but was fully sensitive to inhibition at 23 degrees C. Furthermore, addition of cyclic AMP to a cell culture medium shortened the period of pheromone-induced G1 arrest. We conclude that inhibition of adenylate cyclase activity by alpha factor may constitute, at least in part, the biochemical mode of action of the pheromone in vivo.

Adenylyl Cyclase Inhibitors↗
PubMed1979

Recovery of S. cerevisiae a cells from G1 arrest by alpha factor pheromone requires endopeptidase action.

Radioactive alpha factor is degraded to discrete biologically inactive fragments by the target a cells of S. cerevisiae, but not by alpha cells which make the pheromone. The pattern of cleavage products and sequence analysis of one fragment indicated that the first scission occurred between leucine 6 and lysine 7. The protease inhibitors tosyl-L-argininyl-methyl ester (TAME), tosyl-L-lysyl-chloromethylketone (TLCK) and N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin) markedly prolonged the period of G1 arrest in a cells exposed to alpha factor, while other standard protease inhibitors had little or no effect. The presence of TAME and leupeptin, or TLCK, reduced the rate of degradation of radioactively labeled alpha factor by a cells. Intact yeast cells have apparent esterase and amidase activities that are blocked by the same spectrum of inhibitors that potentiate alpha factor action. Purified alpha factor is a competitive inhibitor of these hydrolytic activities. The activities are present in yeast mutants which have greatly reduced levels of the three major vacuole-associated proteases (A, B and C) or which carry an ochre mutation in the major neutral protease (B). These observations indicate that the inactivation of alpha factor is due to endoproteolytic cleavage, the destruction of the pheromone is required to overcome its effects on growth and that degradation of the molecule may involve surface bound endopeptidase(s).

Animals↗
PubMed1978

Identification of tubulin from the yeast Saccharomyces cerevisiae.

A tubulin-like protein was identified in the lower eukaryote Saccharomyces cerevisiae. The following criteria were used: (i) copolymerization of the 35S-labeled yeast protein with porcine brain tubulin; (ii) immunoprecipitation of the 35S-labeled yeast protein with antiflagellar tubulin antibody; (iii) the presence of the yeast protein as a constituent of isolated yeast nuclei; and (iv) splitting of the yeast protein in a gel electrophoretic system containing sodium dodecyl sulfate that resolved the alpha- and beta-tubulin chains from other sources. This protein did not appear to have significant affinity for the plant alkaloid, Colcemid.

Cell Nucleus↗