Yeast Cells In Medium Research Articles

FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast.

FK506-binding protein with a molecular weight of 12 kDa (FKBP12) is a receptor of the immunosuppressive drugs, FK506 and rapamycin. The physiological functions of FKBP12 remain ambiguous because of its nonessentiality and multifunctionality. Here, we show that FKBP12 promotes the utilization of serine as a nitrogen source and regulates the isoleucine biosynthetic pathway in fission yeast. In screening for small molecules that inhibit serine assimilation, we found that the growth of fission yeast cells in medium supplemented with serine as the sole nitrogen source, but not in glutamate-supplemented medium, was suppressed by FKBP12 inhibitors. Knockout of FKBP12 phenocopied the action of these compounds in serine-supplemented medium. Metabolome analyses and genetic screens identified the threonine deaminase, Tda1, to be regulated downstream of FKBP12. Genetic and biochemical analyses unveiled the negative regulation of Tda1 by FKBP12. Our findings reveal new roles of FKBP12 in amino acid biosynthesis and nitrogen metabolism homeostasis.

Artificial induction of cadmium tolerance and its further enhancement via heterologous co-expression of SpHMT1 and AtPCS1 in the yeast cells

Microorganisms can be used in the bioremediation of heavy metals. It is desirable if the microorganisms show a strong tolerance as well as the ability to accumulate (or biosorb) heavy metals. Yeast is well known for having a high capacity of biosorption of heavy metals. Therefore, the present study focused on developing a yeast mutant that has a strong tolerance to cadmium (Cd), a representative toxic metal. The Cd-resistant yeast mutant (CdR) was induced and isolated by growing yeast cells in media containing Cd and gradually increasing the concentration until reaching a possible maximum of 20 mM CdCl2, to which the cells adapted and survived. CdR cells showed stronger tolerance to Cd-induced stress than the control cells. To obtain even higher Cd tolerance in the yeast cells, both Schizosaccharomyces pombe heavy metal tolerance factor 1 (SpHMT1) and Arabidopsis thaliana phytochelatin synthase (AtPCS1) genes were introduced into the CdR cells to be expressed simultaneously. The transformed CdR cells showed higher Cd tolerance than the untransformed CdR cells.

Control of Phospholipid Synthesis by Phosphorylation of the Yeast Lipin Pah1p/Smp2p Mg2+-dependent Phosphatidate Phosphatase

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1delta cells.

Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Storage, Maintenance, and Working with Living Arrays

INTRODUCTIONThe most difficult aspect of using an array approach for a high-throughput study is maintaining the array's viability while avoiding contamination of the plates. This protocol describes three different storage methods for the array. Frozen stocks are the most permanent method and are an absolute requirement for maintaining a record of the array. Unfortunately, because thawing is necessary to access the strains from a 96-well format, the repeated use of the frozen stocks as a source is expected to eventually lead to loss of viability of the stock. (Generating frozen stocks of the array strains individually, as well as in the arrayed format, is essential.) Maintenance of a water stock provides a source that is more convenient and (relative to yeast cells in media) semi-permanent (~6 months). However, the water stock is fairly precarious because cross-transfer of strains between wells can occur quite easily if the plates are dropped or disturbed. The third method of storage, that of maintaining extra copies of the solid medium array plates, is the most convenient, but it is also the least stable. In short, the best option is to make several frozen stocks. For greater convenience in repeated use of the array, generate water stocks and additional copies of the solid medium plates.

Long-Chain Acyl-CoA–Dependent Regulation of Gene Expression in Bacteria, Yeast and Mammals

Fatty acyl-CoA thioesters are essential intermediates in lipid metabolism. For many years there have been numerous conflicting reports concerning the possibility that these compounds also serve regulatory functions. In this review, we examine the evidence that long-chain acyl-CoA is a regulatory signal that modulates gene expression. In the bacteria Escherichia coli, long-chain fatty acyl-CoA bind directly to the transcription factor FadR. Acyl-CoA binding renders the protein incapable of binding DNA, thus preventing transcription activation and repression of many genes and operons. In the yeast Saccharomyces cerevisiae, genes encoding peroxisomal proteins are activated in response to exogenously supplied fatty acids. In contrast, growth of yeast cells in media containing exogenous fatty acids results in repression of a number of genes, including that encoding the delta9-fatty acid desaturase (OLE1). Both repression and activation are dependent upon the function of either of the acyl-CoA synthetases Faa1p or Faa4p. In mammals, purified hepatocyte nuclear transcription factor 4alpha (HNF-4alpha) like E. coli FadR, binds long chain acyl-CoA directly. Coexpression of HNF-4alpha and acyl-CoA synthetase increases the activation of transcription of a fatty acid-responsive promoter, whereas coexpression with thioesterase decreases the fatty acid-mediated response. Conflicting data exist in support of the notion that fatty acyl-CoA are natural ligands for peroxisomal proliferator-activated receptor alpha (PPARalpha).

Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine

Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae, chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.

Induction of respiration-deficient mutants inSaccharomyces cerevisiaeby chelerythrine

Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae, chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.

ACR1, a gene encoding a protein related to mitochondrial carriers, is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae.

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.

Ofloxacin induces cytoplasmic respiration-deficient mutants in yeast Saccharomyces cerevisiae.

Ofloxacin, a new quinolone with potent antibacterial activity, was also found to be effective against yeast. At relatively high concentrations, and at mild alkaline pH, ofloxacin inhibited the growth of yeast cells in medium containing glucose, and prevented growth on glycerol, as carbon and energy source. The cells growing in the presence of ofloxacin exhibited abberrantly budded forms, lost their viability and many of them converted to cytoplasmic respiration-deficient mutants. Induction of mutants was also observed under non-growing conditions. The petite clones analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.

Alcohol dehydrogenase (ADH) in yeasts II. NAD+‐and NADP+‐dependent alcohol dehydrogenases in Saccharomycopsis lipolytica

AbstractIn Sm. lipolytica one NAD+‐dependent and three NADP+‐dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis.The NAD+‐dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long‐chain alcohols (octanol) than with short‐chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+‐dependent ADH band is gel‐electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+‐dependent alcohol dehydrogenases (ADH II–IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+‐dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+‐dependent alcohol dehydrogenases is regulated by the carbon source of the medium.Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allyalcohol in the medium. All isolates of allylalcohol‐resistant mutants show identical growth in medium containing ethanol as the wild type strain.

Alcohol dehydrogenase (ADH) in yeasts. II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica.

In Sm. lipolytica one NAD+-dependent and three NADP+-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD+-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+-dependent alcohol dehydrogenases (ADH II--IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allylalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.

Effect of Copper on Growth of Yeast

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H2S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105,000 g for 60 min.

Studies on influence of copper on microorganisms. I. Effect of copper on growth of yeast.

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H2S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obta...

Biogenesis of mitochondria: 12. The effects of aminoglycoside antibiotics on the mitochondrial and cytoplasmic protein-synthesizing systems of Saccharomyces cerevisiae

The aminoglycoside antibiotics can be divided into two groups on the basis of their effects on Saccharomyces cerevisiae. The first group, consisting of neamine, kanamycin, streptomycin, and viomycin had only minor effects on the growth of yeast cells and on in vitro protein synthesis. The second group, comprising neomycin B, neomycin C, and paromomycin, had complex effects on the growth and survival of yeast cells, and potently inhibited the in vitro incorporation of amino acids into protein by mitochondria and by cytoplasmic ribosomes. The effects of the neomycins and paromomycin on the growth of yeast cells in media containing either fermentable or non-fermentable substrate indicated that in vivo these antibiotics differentiated only marginally between the mitochondrial and cytoplasmic ribosomal protein-synthesising systems. In marked contrast, the incorporation of amino acids by the mitochondrial protein-synthesizing system in vitro was considerably more sensitive to the neomycins and paromomycin than was the cytoplasmic ribosomal system. A probable explanation of this discrepancy is proposed. The mitochondrial protein-synthesizing system of rat liver was compared with that of yeast, and was found to differ in being insensitive to the neomycins and paromomycin. The possible significance of this phylogenetic difference is discussed.