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J Thorner

Publications and source records attributed to J Thorner.

At least 91 records · Page 5Linked to original sources
PubMed1989

Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans.

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay. The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations.

Amino Acid Sequence↗
PubMed1989

Vertebrate and yeast calmodulin, despite significant sequence divergence, are functionally interchangeable.

Yeast strains relying solely on vertebrate (Xenopus laevis) calmodulin, expressed under control of a yeast (GAL1) promoter, grew at the same rate as yeast cells containing their endogenous calmodulin. Therefore, the ability to perform essential functions has been conserved between yeast and vertebrate calmodulins, suggesting that calmodulin performs the same (or overlapping) roles in yeast as it does in higher eukaryotes. Successful substitution of vertebrate for yeast calmodulin also suggests that the two proteins can adopt similar conformations in vivo, despite the large number of amino acid differences between them (60 out of 148 residues). Strains overproducing either vertebrate or yeast calmodulin about 100-fold and a strain producing a normal level of yeast calmodulin were essentially indistinguishable in many characteristics, including microtubule distribution, rate of secretion, response to mating pheromone, sporulation, and adaptation to nutrient limitation. Calmodulin overproduction did not confer elevated resistance to a phenothiazine drug, trifluoperazine, thought to be a calmodulin-specific inhibitor. These results have important implications for understanding the role of calmodulin in intracellular calcium signaling.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease.

The KEX2-encoded endoprotease was overproduced in yeast several hundred-fold and further purified to achieve a 10,000-fold enrichment in specific activity. The enzyme was (i) membrane-bound, but solubilized by detergents; (ii) able to cleave peptide substrates at both Lys-Arg and Arg-Arg sites; (iii) inhibited by EDTA and EGTA (but not o-phenanthroline), but fully reactivated by Ca2+; (iv) unaffected by 5-10 mM phenylmethylsulfonyl fluoride, N alpha-(ptosyl)lysine chloromethyl ketone, or L-1-tosylamido-2-phenylethyl chloromethyl ketone, but inactivated by 1-2 microM Ala-Lys-Arg-chloromethyl ketone; (v) labeled specifically by 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone; and (vi) resistant to trans-epoxysuccinate compounds (which inactivate thiol proteases), but inactivated by diisopropyl fluorophosphate (a diagnostic serine protease inhibitor). Mutant enzyme molecules lacking as many as 200 C-terminal residues still retained Ca2+-dependent protease activity and were labeled by 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone.

Base Sequence↗
PubMed1989

The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements.

The activity and cell-type specificity of the promoter of the MF alpha 1 gene of Saccharomyces cerevisiae were examined by measuring expression of an MF alpha 1-SUC2 gene fusion in MATa, MAT alpha, and MATa/MAT alpha cells. A high level of invertase activity was observed only in MAT alpha cells. Weak expression occurred in MATa cells when the hybrid gene was carried on a multicopy plasmid or on a centromere-containing plasmid, but not when the hybrid gene was integrated at the normal MF alpha 1 locus. Analysis of a set of 5'-deletions of the promoter region of the MF alpha 1-SUC2 gene on the multicopy plasmid indicated that sequences from -354 to -274 upstream of the translational start site were required for high level expression in MAT alpha cells. Smaller internal deletions and insertions within the promoter region of the MF alpha 1-SUC2 gene were inserted into the genome at the normal MF alpha 1 locus. These mutations further delineated four promoter domains important for expression: (1) two 26 bp elements (-365 to -340 and -312 to -287) with imperfect dyad symmetry; (2) a 40 bp segment (-264 to -226) that lies about 120 bp upstream of the TATA box; and (3) the TATA box itself (-128 to -122). The transcriptional start sites of the normal MF alpha 1 promoter and of a mutant lacking the TATA box were determined. The MF alpha 1 locus was mapped to the left arm of chromosome XVI, about 22 cM centromere-proximal to the PEP4 gene.

Base Sequence↗
PubMed1989

Nucleotidylation, not phosphorylation, is the major source of the phosphotyrosine detected in enteric bacteria.

The majority of the phosphotyrosine recovered from partial acid hydrolysates of 32P-labeled Escherichia coli is derived from a single prominent protein. We show here by biochemical, genetic, and immunological criteria that this protein is actually glutamine synthetase adenylylated (not phosphorylated) at tyrosine. Furthermore, all of the phosphotyrosine detectable in partial acid hydrolysates of 32P-labeled Salmonella typhimurium was eliminated in a strain deficient in both glutamine synthetase and uridylyltransferase, an enzyme which uridylylates the regulatory protein PII at a tyrosine residue. These results suggest that protein-tyrosine phosphorylation represents a rare modification in eubacterial cells.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1988

The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain.

The alpha-factor receptor is rapidly hyperphosphorylated on Thr and Ser residues in its hydrophilic C-terminal domain after cells are exposed to pheromone. Mutant receptors in which this domain is altered or removed are biologically active and bind alpha-factor with nearly normal affinity. However, cells expressing the mutant receptors are hypersensitive to pheromone action and appear to be defective in recovery from alpha-factor-induced growth arrest. Mutant receptors with partial C-terminal truncations undergo ligand-induced endocytosis, suggesting that down-regulation of receptor number is not the sole process for adaptation at the receptor level. A mutant receptor lacking the entire C-terminal domain (134 residues) does not display ligand-induced endocytosis. Genetic experiments indicate that the contribution of SST2 function to adaptation does not require the C-terminal domain of the receptor.

Amino Acid Sequence↗
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↗
PubMed1988

Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells.

Mammalian cell lines (BSC-40, NG108-15, and GH4C1) that cannot process the murine neuroendocrine peptide precursor prepro-opiomelanocortin (mPOMC) when its synthesis is directed by a vaccinia virus vector were coinfected with a second recombinant vaccinia virus carrying the yeast KEX2 gene, which encodes an endopeptidase that cleaves at pairs of basic amino acid residues. mPOMC was cleaved intracellularly to a set of product peptides normally found in vivo, including mature gamma-lipotropin and beta-endorphin1-31. In GH4C1 cells (a rat pituitary line), product peptides were incorporated into stored secretory granules. These results suggest that the inability of any particular cell line to process a prohormone precursor is due to the absence of a suitable endogenous processing enzyme.

Cell Line↗
PubMed1987

The yeast ARD1 gene product is required for repression of cryptic mating-type information at the HML locus.

Mutations in the ARD1 gene prevent yeast cells from displaying G1-specific growth arrest in response to nitrogen deprivation and cause MATa haploids (but not MAT alpha haploids) to be mating defective. Analysis of cell type-specific gene expression by examination of RNA transcripts and measurement of beta-galactosidase activity from yeast gene-lacZ fusions demonstrated that the mating defect of MATa ard1 mutants was due to an inability to express genes required by MATa cells for the mating process. The lack of mating-specific gene expression in MATa cells was found to be due solely to derepression of the normally silent alpha information at the HML locus. The cryptic a information at the HMR locus was only very slightly derepressed in ard1 mutants, to a level insufficient to affect the mating efficiency of MAT alpha cells. The preferential elevation of expression from HML over HMR was also observed in ard1 mutants which contained the alternate arrangement of a information at HML and alpha information at HMR. Hence, the effect of the ard1 mutation was position specific (rather than information specific). Although the phenotype of ard1 mutants resembled that of cells with mutations in the SIR1 gene, both genetic and biochemical findings indicated that ARD1 control of HML expression was independent of the regulation imposed by SIR1 and the other SIR genes. These results suggest that the ARD1 gene encodes a protein product that acts, directly or indirectly, at the HML locus to repress its expression and, by analogy, may control expression of other genes involved in monitoring nutritional conditions.

Diploidy↗
PubMed1987

The yeast repeated element sigma contains a hormone-inducible promoter.

A genomic clone (lambda ScG7) from Saccharomyces cerevisiae encoded a 650-nucleotide poly(A)-containing [poly(A)+] RNA that was about 50 times more abundant in MATa cells that had been exposed to the peptide pheromone alpha-factor than in untreated cells. This RNA was transcribed from a cluster of repetitive sequences: both intact and truncated delta and sigma elements adjacent to a tRNATrp gene. Strand-specific probes indicated that this RNA initiated within an intact sigma element and contained sigma sequences at its 5' end. MATa cells produced two other prominent poly(A)+ RNAs (500 and 5,300 bases) in response to alpha-factor that were homologous to the same strand of sigma but transcribed from other locations in the genome. Induction of the sigma-related transcripts was rapid, was not blocked by inhibition of protein synthesis, required a functional receptor (STE2 gene product), and hence appeared to be a primary response to pheromone. Pulse-labeling confirmed that accumulation of sigma RNA following alpha-factor administration was accounted for by an increase in its rate of transcription. The sigma RNAs also were induced in MAT alpha cells that had been treated with a-factor, but were not present at significant levels in MATa/MAT alpha diploids. In MATa cells transformed with a plasmid in which the lambda ScG7 sigma element was inserted just upstream of a gene coding for the intracellular form of invertase (SUC2) lacking its own promoter, a new poly(A)+ RNA (2.2 kilobases) appeared in response to alpha-factor that hybridized to both sigma and SUC2 probes, and intracellular invertase activity was elevated about 10-fold within 30 min. Primer extension showed that transcription from the hybrid gene initiated exclusively within the sigma sequence (117 nucleotides from the 3' end of the element).

Chromosome Mapping↗
PubMed1986

Isolation of the yeast calmodulin gene: calmodulin is an essential protein.

Calmodulin was purified from Saccharomyces cerevisiae based on its characteristic properties. Like other calmodulins, the yeast protein is small, heat-stable, acidic, retained by hydrophobic matrices in a Ca2+-dependent manner, exhibits a pronounced Ca2+-induced shift in electrophoretic mobility, and binds 45Ca2+. Using synthetic oligonucleotide probes designed from the sequences of two tryptic peptides derived from the purified protein, the gene encoding yeast calmodulin was isolated. The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II. The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins. Disruption or deletion of the yeast calmodulin gene results in a recessive-lethal mutation; thus, calmodulin is essential for the growth of yeast cells.

Amino Acid Sequence↗
PubMed1986

Protein-tyrosine kinase activity in Saccharomyces cerevisiae.

Saccharomyces cerevisiae was examined for tyrosine kinase activity in vitro because this organism offers molecular and genetic approaches for analyzing the role of tyrosine phosphorylation in cellular growth control that are unavailable in higher eukaryotes. Yeast extracts phosphorylated a random copolymer (glutamic acid:tyrosine, 80:20) at tyrosine in a reaction that was linear with respect to time and protein concentration. In the absence of added copolymer, phosphotyrosine was 0.1 percent of the total phosphoamino acids labeled with [gamma-32P]adenosine triphosphate in endogenous yeast proteins. However, specific activities of these reactions were low (approximately 1 percent of those in extracts of chick embryo fibroblasts). Lack of significant incorporation of label from [alpha-32P]adenosine triphosphate into the copolymer or endogenous yeast proteins demonstrated that nucleotide interconversion, adenylylation, and subsequent hydrolysis could not account for the generation of phosphotyrosine observed.

Intercellular Signaling Peptides and Proteins↗
PubMed1986

Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene.

The structural gene for alpha-aminoadipate reductase (LYS2) was isolated from a Saccharomyces cerevisiae genomic DNA library by complementation of a lys2 mutant. Both genetic and biochemical criteria confirmed that the DNA obtained corresponds to the LYS2 locus on chromosome II. Subcloning and deletion analysis showed that a functional LYS2 gene is contained within a 4.6-kilobase (kb) EcoRI-HindIII fragment of the original insert, and the slightly larger EcoRI-ClaI segment (4.8 kb) was used to construct a series of cloning vehicles, including integrating, episomal, replicative, and centromeric vectors. The cloned DNA was also used to generate a genomic deletion that lacks all LYS2 coding sequences on chromosome II. The level of the LYS2 transcript (4.2 kb) was 10-fold higher in cells grown on minimal medium than in cells grown on complete medium and was not repressed by the presence of lysine alone. Gene disruption, gene replacement, and promoter analysis of the major alpha-factor structural gene (MF alpha 1) were performed to illustrate the utility of the LYS2 gene for the genetic manipulation of yeasts. Because all fungi synthesize lysine via the alpha-aminoadipate pathway, the techniques developed here for using the S. cerevisiae LYS2 gene should be directly applicable to other fungal systems.

Aldehyde Oxidoreductases↗