Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin. (65/402)

We have identified two Saccharomyces cerevisiae genes that, in high copy, confer resistance to Kluyveromyces lactis zymocin, an inhibitor that blocks cells in the G(1) phase of the cell cycle prior to budding and DNA replication. One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase. Increased SAP155 dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the four SAP genes in conferring zymocin resistance in high copy, but is antagonized by high-copy SAP185 or SAP190. Since cells lacking SIT4 or deleted for both SAP185 and SAP190 are also zymocin resistant, our data support a model whereby high-copy SAP155 promotes resistance by competition with the endogenous levels of SAP185 and SAP190 expression. Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase. Mutations affecting the RNA polymerase II Elongator complex also confer K. lactis zymocin resistance. Since sit4Delta and SAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.  (+info)

Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis. (66/402)

Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for the expression of heterologous proteins. An anti-Ras single-chain antibody fragment (scFv) coding sequence was fused in-frame to different pre- or prepro-regions, or downstream from a reporter secretory gene (Arxula adeninivorans glucoamylase), separated by a Kex2 protease (Kex2p)-like processing sequence. Both organisms are able to secrete soluble scFv, with yields depending on the nature of the expression cassette, up to levels ranging from 10 to 20 mg l(-1). N-terminal sequence analysis of the purified scFv showed that fusions are correctly processed to the mature scFv by a signal peptidase or a Kex2p-type endoprotease present in Y. lipolytica and K. lactis. The scFv protein also retains the capacity to bind to a glutathioneS-transferase (GST)-Harvey-Ras(Val12) fusion, indicating that the antibody is functional. These results indicate that the yeasts Y. lipolytica and K. lactis have potential for industrial production of soluble and active scFv.  (+info)

Dynamics of telomeric DNA turnover in yeast. (67/402)

Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.  (+info)

Saccharomyces cerevisiae RNA polymerase II is affected by Kluyveromyces lactis zymocin. (68/402)

The G(1) arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae cells requires a functional RNA polymerase II (pol II) Elongator complex. In studying a link between zymocin and pol II, progressively truncating the carboxyl-terminal domain (CTD) of pol II was found to result in zymocin hypersensitivity as did mutations in four different CTD kinase genes. Consistent with the notion that Elongator preferentially associates with hyperphosphorylated (II0) rather than hypophosphorylated (IIA) pol II, the II0/IIA ratio was imbalanced toward II0 on zymocin treatment and suggests zymocin affects pol II function, presumably in an Elongator-dependent manner. As judged from chromatin immunoprecipitations, zymocin-arrested cells were affected with regards to pol II binding to the ADH1 promotor and pol II transcription of the ADH1 gene. Thus, zymocin may interfere with pol II recycling, a scenario assumed to lead to down-regulation of pol II transcription and eventually causing the observed G(1) arrest.  (+info)

Recombinational telomere elongation promoted by DNA circles. (69/402)

Yeast mutants lacking telomerase are capable of maintaining telomeres by an alternate mechanism that depends on homologous recombination. We show here, by using Kluyveromyces lactis cells containing two types of telomeric repeats, that recombinational telomere elongation generates a repeating pattern common in most or all telomeres in survivors that retain both repeat types. We propose that these patterns arise from small circles of telomeric DNA being used as templates for rolling-circle gene conversion and that the sequence from the lengthened telomere is spread to other telomeres by additional, more typical gene conversion events. Consistent with this, artificially constructed circles of DNA containing telomeric repeats form long tandem arrays at telomeres when transformed into K. lactis cells. Mixing experiments done with two species of telomeric circles indicated that all of the integrated copies of the transforming sequence arise from a single original circular molecule.  (+info)

Pyruvate decarboxylase from Kluyveromyces lactis. An enzyme with an extraordinary substrate activation behaviour. (70/402)

Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady-state kinetics, stopped-flow measurements, and kinetic dilution experiments. The dependence of the observed activation rate constant on the substrate concentration of pyruvate decarboxylase from K. lactis showed a minimum at a pyruvate concentration of 1.5 mm. According to the mechanism of substrate activation suggested this local minimum occurs due to the big ratio of the dissociation constants for the binding of the first (regulatory) and the second (catalytic) substrate molecule. The microscopic rate constants of the substrate activation could be determined by a refined fit procedure. The influence of the artificial activator pyruvamide on the activation of the enzyme was studied.  (+info)

Gene order evolution and paleopolyploidy in hemiascomycete yeasts. (71/402)

The wealth of comparative genomics data from yeast species allows the molecular evolution of these eukaryotes to be studied in great detail. We used "proximity plots" to visually compare chromosomal gene order information from 14 hemiascomycetes, including the recent Genolevures survey, to Saccharomyces cerevisiae. Contrary to the original reports, we find that the Genolevures data strongly support the hypothesis that S. cerevisiae is a degenerate polyploid. Using gene order information alone, 70% of the S. cerevisiae genome can be mapped into "sister" regions that tile together with almost no overlap. This map confirms and extends the map of sister regions that we constructed previously by using duplicated genes, an independent source of information. Combining gene order and gene duplication data assigns essentially the whole genome into sister regions, the largest gap being only 36 genes long. The 16 centromere regions of S. cerevisiae form eight pairs, indicating that an ancestor with eight chromosomes underwent complete doubling; alternatives such as segmental duplications can be ruled out. Gene arrangements in Kluyveromyces lactis and four other species agree quantitatively with what would be expected if they diverged from S. cerevisiae before its polyploidization. In contrast, Saccharomyces exiguus, Saccharomyces servazzii, and Candida glabrata show higher levels of gene adjacency conservation, and more cases of imperfect conservation, suggesting that they split from the S. cerevisiae lineage after polyploidization. This finding is confirmed by sequences around the C. glabrata TRP1 and IPP1 loci, which show that it contains sister regions derived from the same duplication event as that of S. cerevisiae.  (+info)

Trehalose-mediated inhibition of the plasma membrane H+-ATPase from Kluyveromyces lactis: dependence on viscosity and temperature. (72/402)

The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures. At 20 degrees C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased V(max) and increased S(0.5) (substrate concentration when initial velocity equals 0.5 V(max)), mainly at high trehalose concentrations (0.6 to 0.8 M). The quotient V(max)/S(0.5) decreased from 5.76 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the absence of trehalose to 1.63 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the presence of 0.8 M trehalose. The decrease in V(max) was linearly dependent on solution viscosity (eta), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramer's theory for reactions in solution. In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted. In the absence of trehalose, increasing the temperature up to 40 degrees C resulted in an exponential increase in V(max) and a decrease in enzyme cooperativity (n), while S(0.5) was not modified. As temperature increased, the effect of trehalose on V(max) decreased to become negligible at 40 degrees C, in good correlation with the temperature-mediated decrease in viscosity. The trehalose-mediated increase in S(0.5) was similar at all temperatures tested, and thus, trehalose effects on V(max)/S(0.5) were always observed. Trehalose increased the activation energy for ATP hydrolysis. Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.  (+info)