VDAC contributes to mRNA levels in Saccharomyces cerevisiae cells by the intracellular reduction/oxidation state dependent and independent mechanisms

The PUT2 gene was isolated on a 6.5-kilobase insert of a recombinant DNA plasmid by functional complementation of a put2 (delta 1-pyrroline-5-carboxylate dehydrogenase-deficient) mutation in Saccharomyces cerevisiae. Its identity was confirmed by a gene disruption technique in which the chromosomal PUT2+ gene was replaced by plasmid DNA carrying the put2 gene into which the S. cerevisiae HIS3+ gene had been inserted. The cloned PUT2 gene was used to probe specific mRNA levels: full induction of the PUT2 gene resulted in a 15-fold increase over the uninduced level. The PUT2-specific mRNA was approximately 2 kilobases in length and was used in S1 nuclease protection experiments to locate the gene to a 3-kilobase HindIII fragment. When delta 1-pyrroline-5-carboxylate dehydrogenase activity levels were measured in strains carrying the original plasmid, as well as in subclones, similar induction ratios were found as compared with enzyme levels in haploid yeast strains. Effects due to increased copy number or position were also seen. The cloned gene on a 2 mu-containing vector was used to map the PUT2 gene to chromosome VIII.

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Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.2941-2948.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 2941-2948

Author(s):

A Lombardo ◽

G P Cereghino ◽

I E Scheffler

Keyword(s):

Saccharomyces Cerevisiae ◽

Succinate Dehydrogenase ◽

Half Life ◽

Glucose Repression ◽

Promoter Sequence ◽

Regulatory Elements ◽

Mrna Levels ◽

Protein Subunit ◽

Gene Encoding ◽

Regulatory Complex

We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae. The gene had been cloned by us and shown to be subject to glucose regulation (A. Lombardo, K. Carine, and I. E. Scheffler, J. Biol. Chem. 265:10419-10423, 1990). We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA. In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min. The gene is also regulated transcriptionally by glucose. The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex. A deletion analysis has shown that the two distal elements are redundant. These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence. These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.

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Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2914-2924.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2914-2924

Author(s):

A Hoekema ◽

R A Kastelein ◽

M Vasser ◽

H A de Boer

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Steady State ◽

Codon Usage ◽

Experimental Approach ◽

Mrna Translation ◽

Yeast Cells ◽

Expression Level ◽

Start Codon ◽

Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

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Oxidation-State-Dependent Binding Properties of the Active Site in a Mo-Containing Formate Dehydrogenase

Journal of the American Chemical Society ◽

10.1021/jacs.7b03958 ◽

2017 ◽

Vol 139 (29) ◽

pp. 9927-9936 ◽

Cited By ~ 31

Author(s):

William E. Robinson ◽

Arnau Bassegoda ◽

Erwin Reisner ◽

Judy Hirst

Keyword(s):

Oxidation State ◽

Active Site ◽

Formate Dehydrogenase ◽

Binding Properties ◽

State Dependent

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The effect of calnexin deletion on the expression level of PDI in Saccharomyces cerevisiae under heat stress conditions

Cellular & Molecular Biology Letters ◽

10.2478/s11658-007-0033-y ◽

2008 ◽

Vol 13 (1) ◽

Cited By ~ 2

Author(s):

Huili Zhang ◽

Jianwei He ◽

Yanyan Ji ◽

Akio Kato ◽

Youtao Song

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rate ◽

Heat Stress ◽

Protein Expression ◽

Molecular Chaperone ◽

Mrna Level ◽

Stress Conditions ◽

Mrna Levels ◽

Wild Type ◽

Wild Type Yeast

AbstractWe cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37°C, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.

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Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3992-3998.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3992-3998

Author(s):

A M Dranginis

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Yeast Cells ◽

Specific Gene ◽

Activity Levels ◽

Sporulation Medium ◽

Mrna Levels ◽

Yeast Saccharomyces Cerevisiae ◽

Type Locus ◽

Diploid Cells

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

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GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.4135-4144.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 4135-4144

Author(s):

J Albertyn ◽

S Hohmann ◽

J M Thevelein ◽

B A Prior

Keyword(s):

Saccharomyces Cerevisiae ◽

Osmotic Stress ◽

Yeast Cells ◽

Mrna Levels ◽

Hog Pathway ◽

Hypertonic Medium ◽

High Osmolarity ◽

High Osmolarity Glycerol ◽

Enhanced Production ◽

Glycerol 3 Phosphate Dehydrogenase

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.

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Identification and Characterization ofMAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

Journal of Bacteriology ◽

10.1128/jb.180.11.2875-2882.1998 ◽

1998 ◽

Vol 180 (11) ◽

pp. 2875-2882 ◽

Cited By ~ 101

Author(s):

Eckhard Boles ◽

Patricia de Jong-Gubbels ◽

Jack T. Pronk

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activity ◽

Pyruvate Kinase ◽

Malic Enzyme ◽

Fold Increase ◽

Growth Conditions ◽

Anaerobic Growth ◽

Oxidative Decarboxylation ◽

Mrna Levels ◽

Malic Enzyme Activity

ABSTRACT Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

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The Saccharomyces cerevisiae ICL2 Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism

Journal of Bacteriology ◽

10.1128/jb.182.24.7007-7013.2000 ◽

2000 ◽

Vol 182 (24) ◽

pp. 7007-7013 ◽

Cited By ~ 72

Author(s):

Marijke A. H. Luttik ◽

Peter Kötter ◽

Florian A. Salomons ◽

Ida J. van der Klei ◽

Johannes P. van Dijken ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Nitrogen Source ◽

Coenzyme A ◽

Isocitrate Lyase ◽

Significant Activity ◽

Mrna Levels ◽

Wild Type ◽

Cell Extracts ◽

Lyase Activity ◽

Null Mutants

ABSTRACT The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologousICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced, ICL2 cannot complement the growth defect oficl1 null mutants. It has therefore been suggested thatICL2 encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whetherICL2 encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-type S. cerevisiae and of isogenicicl1, icl2, and icl1 icl2 null mutants. Isocitrate lyase activity was absent in icl1 andicl1 icl2 null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and single icl mutants but not in the icl1 icl2mutant. This demonstrated that ICL2 encodes a specific 2-methylisocitrate lyase and that the ICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that theICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that of ICL1, transcription of ICL2 is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction ofICL2 mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.

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