962 resultados para FERMENTING YEAST
Resumo:
Yeast phosphatidylinositol transfer protein (Sec14p) function is essential for production of Golgi-derived secretory vesicles, and this requirement is bypassed by mutations in at least seven genes. Analyses of such ‘bypass Sec14p’ mutants suggest that Sec14p acts to maintain an essential Golgi membrane diacylglycerol (DAG) pool that somehow acts to promote Golgi secretory function. SPO14 encodes the sole yeast phosphatidylinositol-4,5-bisphosphate-activated phospholipase D (PLD). PLD function, while essential for meiosis, is dispensable for vegetative growth. Herein, we report specific physiological circumstances under which an unanticipated requirement for PLD activity in yeast vegetative Golgi secretory function is revealed. This PLD involvement is essential in ‘bypass Sec14p’ mutants where normally Sec14p-dependent Golgi secretory reactions are occurring in a Sec14p-independent manner. PLD catalytic activity is necessary but not sufficient for ‘bypass Sec14p’, and yeast operating under ‘bypass Sec14p’ conditions are ethanol-sensitive. These data suggest that PLD supports ‘bypass Sec14p’ by generating a phosphatidic acid pool that is somehow utilized in supporting yeast Golgi secretory function.
Resumo:
Telomere length is maintained through a dynamic balance between addition and loss of the terminal telomeric DNA. Normal telomere length regulation requires telomerase as well as a telomeric protein–DNA complex. Previous work has provided evidence that in the budding yeasts Kluyveromyces lactis and Saccharomyces cerevisiae, the telomeric double-stranded DNA binding protein Rap1p negatively regulates telomere length, in part by nucleating, by its C-terminal tail, a higher-order DNA binding protein complex that presumably limits access of telomerase to the chromosome end. Here we show that in K. lactis, truncating the Rap1p C-terminal tail (Rap1p-ΔC mutant) accelerates telomeric repeat turnover in the distal region of the telomere. In addition, combining the rap1-ΔC mutation with a telomerase template mutation (ter1-kpn), which directs the addition of mutated telomeric DNA repeats to telomeres, synergistically caused an immediate loss of telomere length regulation. Capping of the unregulated telomeres of these double mutants with functionally wild-type repeats restored telomere length control. We propose that the rate of terminal telomere turnover is controlled by Rap1p specifically through its interactions with the most distal telomeric repeats.
Resumo:
The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active β subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.
Resumo:
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two types of subunits: RNR1 contains the active site for reduction and the binding sites for the nucleotide allosteric effectors. RNR2 contains the diiron-tyrosyl radical (Y⋅) cofactor essential for the reduction process. Studies in yeast have recently identified four RNR subunits: Y1 and Y3, Y2 and Y4. These proteins have been expressed in Saccharomyces cerevisiae and in Escherichia coli and purified to ≈90% homogeneity. The specific activity of Y1 isolated from yeast and E. coli is 0.03 μmol⋅min−1⋅mg−1 and of (His)6-Y2 [(His)6-Y2-K387N] from yeast is 0.037 μmol⋅min−1⋅mg−1 (0.125 μmol⋅min−1⋅mg−1). Y2, Y3, and Y4 isolated from E. coli have no measurable activity. Efforts to generate Y⋅ in Y2 or Y4 using Fe2+, O2, and reductant have been unsuccessful. However, preliminary studies show that incubation of Y4 and Fe2+ with inactive E. coli Y2 followed by addition of O2 generates Y2 with a specific activity of 0.069 μmol⋅min−1⋅mg−1 and a Y⋅. A similar experiment with (His)6-Y2-K387N, Y4, O2, and Fe2+ results in an increase in its specific activity to 0.30 μmol⋅min−1⋅mg−1. Studies with antibodies to Y4 and Y2 reveal that they can form a complex in vivo. Y4 appears to play an important role in diiron-Y⋅ assembly of Y2.
Resumo:
Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.
Resumo:
Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.
Resumo:
A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.
Resumo:
To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [γ-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.
Resumo:
A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (“endoreplication”) or initiation of mitosis before DNA is fully replicated (“mitotic catastrophe”). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of “Start” control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1− (size control at Start), cdc13Δ and rum1OP (endoreplication), and wee1− rum1Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.
Resumo:
Telomerase is an RNA-directed DNA polymerase, composed of RNA and protein subunits, that replicates the telomere ends of linear eukaryotic chromosomes. Using a genetic strategy described here, we identify the product of the EST2 gene, Est2p, as a subunit of telomerase in the yeast Saccharomyces cerevisiae. Est2p is required for enzyme catalysis, as mutations in EST2 were found to result in the absence of telomerase activity. Immunochemical experiments show that Est2p is an integral subunit of the telomerase enzyme. Critical catalytic residues present in RNA-directed DNA polymerases are conserved in Est2p; mutation of one such residue abolishes telomerase activity, suggesting a direct catalytic role for Est2p.
Resumo:
A gene homologous to methionine sulfoxide reductase (msrA) was identified as the predicted ORF (cosmid 9379) in chromosome V of Saccharomyces cerevisiae encoding a protein of 184 amino acids. The corresponding protein has been expressed in Escherichia coli and purified to homogeneity. The recombinant yeast MsrA possessed the same substrate specificity as the other known MsrA enzymes from mammalian and bacterial cells. Interruption of the yeast gene resulted in a null mutant, ΔmsrA::URA3 strain, which totally lost its cellular MsrA activity and was shown to be more sensitive to oxidative stress in comparison to its wild-type parent strain. Furthermore, high levels of free and protein-bound methionine sulfoxide were detected in extracts of msrA mutant cells relative to their wild-type parent cells, under various oxidative stresses. These findings show that MsrA is responsible for the reduction of methionine sulfoxide in vivo as well as in vitro in eukaryotic cells. Also, the results support the proposition that MsrA possess an antioxidant function. The ability of MsrA to repair oxidative damage in vivo may be of singular importance if methionine residues serve as antioxidants.
Resumo:
Sequence divergence acts as a potent barrier to homologous recombination; much of this barrier derives from an antirecombination activity exerted by mismatch repair proteins. An inverted repeat assay system with recombination substrates ranging in identity from 74% to 100% has been used to define the relationship between sequence divergence and the rate of mitotic crossing-over in yeast. To elucidate the role of the mismatch repair machinery in regulating recombination between mismatched substrates, we performed experiments in both wild-type and mismatch repair defective strains. We find that a single mismatch is sufficient to inhibit recombination between otherwise identical sequences, and that this inhibition is dependent on the mismatch repair system. Additional mismatches have a cumulative negative effect on the recombination rate. With sequence divergence of up to approximately 10%, the inhibitory effect of mismatches results mainly from antirecombination activity of the mismatch repair system. With greater levels of divergence, recombination is inefficient even in the absence of mismatch repair activity. In both wild-type and mismatch repair defective strains, an approximate log-linear relationship is observed between the recombination rate and the level of sequence divergence.
Resumo:
Spores harboring an ACC1 deletion derived from a diploid Saccharomyces cerevisiae strain, in which one copy of the entire ACC1 gene is replaced with a LEU2 cassette, fail to grow. A chimeric gene consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat cytosolic acetyl-CoA carboxylase (ACCase) cDNA, and yeast ACC1 3′ tail was used to complement a yeast ACC1 mutation. The complementation demonstrates that active wheat ACCase can be produced in yeast. At low concentrations of galactose, the activity of the “wheat gene” driven by the GAL10 promoter is low and ACCase becomes limiting for growth, a condition expected to enhance transgenic yeast sensitivity to wheat ACCase-specific inhibitors. An aryloxyphenoxypropionate and two cyclohexanediones do not inhibit growth of haploid yeast strains containing the yeast ACC1 gene, but one cyclohexanedione inhibits growth of the gene-replacement strains at concentrations below 0.2 mM. In vitro, the activity of wheat cytosolic ACCase produced by the gene-replacement yeast strain is inhibited by haloxyfop and cethoxydim at concentrations above 0.02 mM. The activity of yeast ACCase is less affected. The wheat plastid ACCase in wheat germ extract is inhibited by all three herbicides at concentrations below 0.02 mM. Yeast gene-replacement strains will provide a convenient system for the study of plant ACCases.
Resumo:
The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.
Resumo:
Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14α-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14α-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.