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At least 1,243 records · Page 69Linked to original sources
PubMed1999

Potato StubSNF1 interacts with StubGAL83: a plant protein kinase complex with yeast and mammalian counterparts.

StubSNF1 is a potato cDNA that encodes a protein kinase similar to the yeast SNF1 gene involved in transcriptional regulation of glucose-repressible genes. The yeast SNF1 functions in a complex with GAL83/SIP1/SIP2 and SNF4 proteins. We have used StubSNF1 as bait in a yeast two-hybrid system to screen for potato cDNAs encoding proteins that bind to StubSNF1. Three overlapping cDNAs, two different in size, were isolated. DNA sequence analysis revealed that they were orthologues of the yeast GAL83/SIP1/SIP2 genes and their mammalian counterparts, AMPK beta-subunits. The direct interaction between the potato proteins StubGAL83 and StubSNF1 was shown by an in vitro binding assay. Southern and Northern hybridisations revealed that StubGAL83 exists in a low copy number in the potato genome and is highly (but organ-specifically) expressed in potato. In contrast, StubSNF1 possesses low transcript levels in each organ, except in flowers where high amounts of StubSNF1 mRNA could be detected. We demonstrate here that StubGAL83 can also interact with yeast SNF4 in a yeast two-hybrid system suggesting that plant SNF1 kinases may function in complexes similar to those detected in yeast and mammals.

Amino Acid Sequence↗
PubMed2000

Complementation of deletion mutants in the genes encoding the F1-ATPase by expression of the corresponding bovine subunits in yeast S. cerevisiae.

The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the inner membrane of the mitochondrion. Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme. The F1-ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of alpha3beta3gammadeltaepsilon. This study demonstrates that four of the five bovine subunits of the F1-ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits. In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains. All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the gamma-, delta- or epsilon-gene became completely, or at a high frequency, cytoplasmically petite. The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide. Expression of the bovine alpha-, beta-, gamma-, and epsilon-, but not the delta-, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes. These results indicate that yeast is able to import, assemble subunits of bovine F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex.

Animals↗
PubMed2001

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast.

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.

Amino Acid Sequence↗
PubMed2001

Comparison of fermentative capacities of industrial baking and wild-type yeasts of the species Saccharomyces cerevisiae in different sugar media.

AIMS: To compare the fermentative capacity of wild and domesticated isolates of the genus Saccharomyces. METHODS AND RESULTS: The fermentative capacity of yeasts from a variety of wild and domesticated sources was tested in synthetic dough media that mimic major bread dough types. Domesticated yeast strains were found to have better maltose-utilizing capacity than wild yeast strains. The capacity to ferment sugars under high osmotic stress was randomly distributed amongst wild and baking strains of Saccharomyces. CONCLUSION: The domestication of bakers' yeast has enhanced the ability of yeasts to ferment maltose, without a similar impact on the fermentative capacity under high osmotic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, combined with molecular studies of both wild and domesticated yeast, showed that domestication of bakers' yeast has resulted in improved maltose utilization, apparently via the duplication and mutation of the MAL genes.

Bread↗
PubMed2001

Effect of selected natural antimicrobials on Baker's yeast activity.

AIMS: To evaluate the responses of Baker's yeast (Saccharomyces cerevisiae) activity to the natural antimicrobials acetic acid, calcium lactate, a lactate-containing cocktail and lactic acid compared to calcium propionate. METHODS AND RESULTS: A dough fermentometer test was used to measure Baker's yeast activity in the presence of these natural antimicrobials and calcium propionate. Yeast activity generally decreased as a function of increasing antimicrobial concentrations, but the lactate-containing cocktail showed no relationship between concentration and yeast activity reduction. At in situ concentrations, calcium propionate resulted in the highest yeast activity reduction, followed by calcium lactate, acetic acid, the lactate-containing cocktail and lactic acid in decreasing order. CONCLUSION: Based on yeast activity reduction, all natural antimicrobials tested showed potential as possible replacements for calcium propionate. SIGNIFICANCE AND IMPACT OF THE STUDY: This has practical implications since calcium propionate inhibits Baker's yeast activity and attracts negative consumer perceptions as a chemical bread preservative.

Acetic Acid↗
PubMed2001

The effects of Malassezia yeasts on cytokine production by human keratinocytes.

Yeasts of Malassezia, members of the microbiologic flora of the skin, cause pityriasis versicolor and have also been implicated in the pathogenesis of other superficial dermatoses; the most important ones are seborrheic dermatitis, folliculitis, and atopic dermatitis. The mechanisms by which the yeasts cause these dermatoseş however, are not yet clear, and there have been no studies on the interaction between fungi and keratinocytes, especially the effects of fungi on the production of cytokines by human keratinocytes. Recently, the genus Malassezia has been expanded to seven species based on molecular data. In this study, we estimated the effects of Malassezia yeasts on cytokine (interleukins 1beta, 6, and 8, monocyte chemotactic protein-1, and tumor necrosis factor-alpha) production by human keratinocytes in order to examine whether the pathogenicity of the respective Malassezia yeasts is different from each other and to elucidate the mechanism by which Malassezia yeasts cause the dermatoses with different clinical and pathologic manifestations. Variable levels of interleukin 6 and 8, and tumor necrosis factor-alpha in the supernatants in response to Malassezia yeasts (except M. furfur) increased from 1 to 24 h co-culture, but the monocyte chemotactic protein-1 was undetectable. Furthermore, cytokine levels in the supernatants were undetectable 1-24 h after the keratinocytes were harvested with only supernatants of Malassezia. These results indicate that Malassezia stimulates cytokine production by keratinocytes, the cytokine production needs the presence of Malassezia, and there are differences in ability to induce cytokine production by human keratinocytes among Malassezia yeasts. These differences may reflect the different inflammatory responses in Malassezia-associated dermatoses, resulting in different clinical and pathologic manifestations.

Cell Survival↗
PubMed1993

Effect of a viable yeast culture on digestibility and rumen fermentation in sheep fed different types of diets.

Five mature wethers fitted with rumen fistulas were fed grass hay and a sugarbeet-pulp-based concentrate or maize silage and a cereal-based concentrate (50/50 digestible organic matter basis), or without with 5 g yeast supplement (Saccharomyces cerevisiae, Biosaf) per day in a latin square design. Diets were given for a 28-d adaptation period, followed by a 10-d collection period to determine digestibility and nitrogen retention data. Afterwards, rumen samples were taken on 3 consecutive days and analysed for volatile fatty acids, pH and ammonia. Digestibility and nitrogen balance were not affected by yeast treatment. Supplementation of yeast increased acetate: propionate ratio, butyrate, isoacids, pH and ammonia. The effects were more pronounced for the maize silage diet. These results demonstrate that the effect of yeast culture on rumen fermentation may depend on the nature of the diet. Living yeast cell number in the rumen fluid rapidly declined when dietary yeast was ceased. Furthermore, yeast cells survived the passage through the digestive tract.

Acetates↗
PubMed2005

Evolution of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes in the yeast Saccharomyces cerevisiae.

The shikimate pathway resulting in three aromatic amino acids is initiated in different organisms by two and three 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases, respectively. Aro3p and Aro4p are the yeast enzymes feedback-inhibited by phenylalanine and tyrosine, respectively. A yeast strain deficient in the general control transcriptional regulatory system of amino acid biosynthesis is unable to live in the presence of high amounts of phenylalanine and tyrosine. Here, we show that this yeast strain can be rescued by the expression of aroH from Escherichia coli encoding the tryptophan-regulated AroH as third isoenzyme. Yeast carrying Ec AroH as the only enzyme for the initial step of the shikimate pathway can grow in the absence of tryptophan. Without aromatic amino acids, this yeast strain survives only when the yeast ARO3 promoter instead of the ARO4 promoter drives E. coli aroH. The detailed analysis of Aro3p and Aro4p revealed a triple feedback control by tyrosine/phenylalanine and tryptophan. Dissecting this control allowed engineering of Aro4p S195A as an enzyme, which is inhibited like AroH only by tryptophan. In addition, Aro4p variants were constructed that show an equally strong inhibition by tyrosine and tryptophan (Aro4p P165G Q302R) and in which the regulation by tyrosine and tryptophan was reversed (Aro4p P165G). Our data suggest that yeast possesses only two instead of three isogenes encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases because both isoenzymes can be fine tuned by tryptophan as additional effector and because transcriptional regulation by the general control system can be induced as backup when aromatic amino acids in the environment are imbalanced.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1976

Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.

Homogeneous RNA polymerase III (RNA nucleotidyltransferase III) has been obtained from yeast. The subunit composition of the enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is composed of 12 putative subunits with molecular weights 160,000, 128,000, 82,000, 41,000, 40,500, 37,000, 34,000, 28,000, 24,000, 20,000, 14,500, and 11,000. The high-molecular-weight subunits and several of the smaller subunits of yeast RNA polymerase III are clearly different from those of enzymes I and II, indicating a distinct molecular structure. However, the molecular weights of some of the small subunits (41,000, 28,000, 24,000, and 14,500) appear to be identical to those of polymerases I and II. Thus, it is possible that the three classes of enzymes in yeast have some common subunits. As in other eukaryotes, yeast polymerase II is inhibited by relatively low concentrations of alpha-amanitin; however, contrary to what has been found in higher eukaryotes, yeast polymerase III is resistant (up to 2 mg/ml) to alpha-amanitin, while yeast polymerase I is sensitive to high concentrations of the drug (50% inhibition at 0.3 mg/ml). These results establish the existence of RNA polymerase III in yeast and provide a structural basis for the discrimination of the three functional polymerases in eukaryotes.

Amanitins↗
PubMed1976

Transcription in yeast: alpha-amanitin sensitivity and other properties which distinguish between RNA polymerases I and III.

Three peaks of DNA-dependent RNA polymerase (RNA nucleotidyltransferase) activity are resolved by chromatography of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named RNA polymerases I, II, and III in order of elution, show similar catalytic properties to the vertebrate class I, class II, and class III RNA polymerases, respectively. Yeast RNA polymerase III is readily distinguished from yeast polymerase I by its biphasic amnonium sulfate activation profile with native DNA templates, greater enzymatic activity with poly[d(I-C)] than with native salmon sperm DNA, and distinctive chromatographic elution positions from DEAE-cellulose (0.12 M ammonium sulfate) compared with DEAE-Sephadex (0.32 M ammonium sulfate). The three yeast RNA polymerases also show significant differences in alpha-amanitin inhibition. RNA polymerase II is the most sensitive (50% inhibition at 1.0 mug of alpha-amanitin per ml). Contrary to the results for vertebrate systems, yeast polymerase I can be completely inhibited by alpha-amanitin at high concentrations (50% inhibition at 600 mug/ml) while yeast RNA polymerase II BEGINS TO SHOW SIGNIFICANT INHIBITION ONLY AT CONCENTRATIONS EXCEEDING 1 MG/ML. Therefore, yeast RNA polymerases I and III show a pattern of alpha-amanitin sensitivity that is the reverse of that seen for the analogous vertebrate RNA polymerases.

Amanitins↗
PubMed1981

Larval selectivity for yeast species by Drosophila mojavensis in natural substrates.

The yeast flora found in the major substrates of Drosophila mojavensis and in larval guts was studied both qualitatively and quantitatively. Quantitative studies show that, in four of the five substrates tested, the larvae did not contain a random sample of the yeasts present in the substrates. One widely distributed cactus yeast, Pichia cactophila, was typically in greater frequency in the larvae than in the substrates. Another cactus yeast, Candida sonorensis, typically exhibited the opposite relationship. Laboratory tests support larval preference behavior rather than differential digestion as being primarily responsible. Larvae are capable of distinguishing between patches of different yeast species and spend more time in patches of preferred yeasts. D. mojavensis appear to be ecological (host plants) generalists and physiological (yeasts) specialists in contrast to the other cactophilic Drosophila. Selective feeding by D. mojavensis larvae in natural substrates may represent optimal foraging behavior.

Journal Article↗
PubMed1983

Instability of dicentric plasmids in yeast.

Dicentric plasmids containing either two copies of centromere 4 or one copy of centromere 4 and one copy of centromere 3 in the yeast plasmid vector YRp17 were constructed in vitro and introduced into yeast cells by DNA transformation. The resulting colonies were heterogeneous for a mixed population of rearranged plasmids. The rearrangements always involved deletion of one or both centromere sequences originally present on the plasmid. Heterogeneity was due to the continued production of deleted plasmids from a pool of unrearranged dicentric plasmids maintained within some of the yeast cells in the colony. The RAD52 gene product is known to be required for the repair of DNA double-strand breaks in yeast. Transformation of rad52 mutant yeast cells with dicentric plasmids gave rearranged plasmids similar to those observed with RAD+ yeast cells, but the transformation frequency was only 5-10% compared to transformation with monocentric plasmids. Also, the ratio of unrearranged dicentric plasmid to deleted plasmids was greatly reduced in the rad52-transformed cells. These observations are consistent with a model in which centromeric DNA sequences can interact independently with the yeast cell spindle apparatus. Occasional movement of centromeres to opposite poles may result in mechanical breakage of plasmid sequences. Plasmids deleted for one or both centromere sequences can be obtained from these broken molecules and are resistant to further rearrangement.

Centromere↗
PubMed1983

Mitotic and meiotic stability of linear plasmids in yeast.

Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells. However, these plasmids are unstable, being rapidly lost from cells growing without selection. Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome. Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids. Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth. Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis. Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid. Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids. However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids. This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability. These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.

Animals↗
PubMed1987

Characterization of avian and viral p60src proteins expressed in yeast.

Avian and viral p60src proteins were expressed from a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. Both the viral and cellular src proteins produced in yeast cells were myristoylated at their amino termini, as is the case for src proteins expressed in chicken embryo fibroblasts. The viral src protein produced in yeast autophosphorylated at tyrosine-416 in vivo and had approximately the same level of in vitro kinase activity as p60v-src expressed in Rous sarcoma virus-transformed cells. Unlike p60c-src expressed in chicken cells, which is phosphorylated on tyrosine in vivo almost exclusively at tyrosine-527, p60c-src expressed in yeast was phosphorylated 2.5-3 times more at tyrosine-416 than at tyrosine-527. The specific activity of the p60c-src produced in yeast was 2.5-5.0 times higher than that of p60c-src overexpressed from a retroviral vector in chicken cells, implicating the altered state of in vivo phosphorylation in modulation of the in vitro kinase activity. The expression of p60v-src substantially slowed down the growth of the yeast cells, suggesting that phosphorylation of yeast proteins essential for cell growth may have interfered with their proper functioning.

Animals↗
PubMed1989

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes.

We have isolated the Arabidopsis thaliana gene (HMG1) encoding 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; (S)-mevalonate:NAD+ oxido-reductase (CoA-acylating), EC 1.1.1.88], the catalyst of the first committed step in isoprenoid biosynthesis. cDNA copies of the plant gene were identified by hybridization with a short, highly conserved segment of yeast HMG-CoA reductase as probe. DNA sequence analysis reveals that the COOH-terminal domain of the Arabidopsis HMG-CoA reductase (containing the catalytic site of the enzyme) is highly conserved with respect to the yeast, mammalian, and Drosophila enzymes, whereas the membrane-bound amino terminus of the Arabidopsis protein is truncated and lacks the complex membrane-spanning architecture of the yeast and animal reductases. Expression of the Arabidopsis gene from the yeast GAL1 promoter in a yeast mutant lacking HMG-CoA reductase activity suppresses the growth defect of the yeast mutant. Taken together, the sequence similarity to other cloned HMG-CoA reductase genes and the suppression of the yeast hmg- mutant provide strong evidence that the novel Arabidopsis gene we have cloned encodes a functional HMG-CoA reductase enzyme.

Amino Acid Sequence↗
PubMed1991

Glycosylation site binding protein and protein disulfide isomerase are identical and essential for cell viability in yeast.

Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4.1) in a variety of multicellular organisms. We have utilized immunological and biochemical techniques to determine if GSBP and PDI are identical in yeast. Antiserum prepared against yeast GSBP identified in microsomes by its ability to be labeled with a peptide photoaffinity probe was found to recognize PDI purified from yeast. Moreover, this purified yeast PDI was found to be specifically labeled by the photoaffinity probe originally used to identify GSBP in a variety of eukaryotes. On the basis of these observations, we conclude that yeast GSBP and PDI are the same protein. The structure of the yeast PDI gene revealed a product with sequence similarity to higher eukaryotic PDI/GSBP. Disruption of this gene in yeast resulted in a recessive lethal mutation, indicating that PDI/GSBP is required for cell viability.

Affinity Labels↗
PubMed1992

Molecular cloning of the gene for the yeast homolog (ACB) of diazepam binding inhibitor/endozepine/acyl-CoA-binding protein.

Diazepam binding inhibitor (DBI)/endozepine (EP)/acyl-CoA-binding protein (ACBP) is a small, highly conserved protein which has been independently isolated and characterized from different species using several different biological systems. To further investigate the structural and functional properties of this protein, we have cloned the homologous gene for DBI/EP/ACBP from the budding yeast Saccharomyces cerevisiae. The yeast gene contains no introns and encodes a polypeptide of 87 amino acids (including the initiating methionine), identical in length to the human gene product with 48% conservation of amino acid residues. The most highly conserved domain consists of 7 contiguous residues which are identical in all known protein species from yeast, birds, and mammals. This domain has previously been shown to constitute the hydrophobic binding site on DBI/EP/ACBP for acyl-CoA esters and is located within the second helical region of the molecule. Major and minor mRNA species of approximately 520 and 740 nucleotides, respectively, were detected in exponentially growing yeast. Sequences similar to those implicated in the regulation of fatty acid synthesis and beta-oxidation in yeast were detected in the promoter region of the gene. The presence of a highly conserved DBI/EP/ACBP gene in a primitive organism such as yeast provides support for the basic biological role of DBI/EP/ACBP as an acyl-CoA-binding protein and suggests that many of the biological functions attributed to it in higher organisms may result from its ability to interact with acyl-CoA. Hence, we have designated the yeast gene as ACB, for acyl-CoA-binding protein.

Amino Acid Sequence↗
PubMed1996

The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione S-conjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.

ATP Binding Cassette Transporter, Subfamily B, Mem↗