Regulation of Pyruvate Carboxylase Isozyme (PYC1, PYC2) Gene Expression in Saccharomyces cerevisiae during Fermentative and Nonfermentative Growth

Brewster, Neil K.; Val, Dale L.; Walker, M.; Wallace, John C.

doi:10.1006/abbi.1994.1209

Cited by 78 publications

(80 citation statements)

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“…Although it has been reported that interruption of PYC1 results in an aspartate requiring phenotype during growth in ethanol [21], we have consistently found that in our strain each gene could substitute for the other for growth in the carbon sources we tested and that the requirement for aspartate was only observed during growth in repressing carbon sources. Different genetic backgrounds that influence the expression of each of the genes may account for this difference in results.…”

Section: Discussionsupporting

confidence: 52%

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae

Blázquez¹,

Gamo²,

Gancedo³

1995

FEBS Letters

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Section: Discussionsupporting

confidence: 52%

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae

Blázquez¹,

Gamo²,

Gancedo³

1995

FEBS Letters

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“…Huet et al (28) observed that the transfer of S. cerevisiae cells from a glutamate medium to an ammonium medium caused a fivefold increase in the mRNAs corresponding to the PYC1 gene in less than 30 min, whereas a decrease occurred when the transfer was done in the opposite direction. The decrease in mRNA seen in stationary-phase ethanol cultures is reminiscent of the behavior of the PYC2 gene from S. cerevisiae reported by Brewster et al (13). These authors observed a fourfold decrease in the levels of that mRNA after the initial exponential phase of growth.…”

Section: Discussionsupporting

confidence: 69%

Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype

Flores

Gancedo

2005

Eukaryot Cell

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We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.Yarrowia lipolytica is a nonconventional yeast that has attracted great attention due to its capacity to grow on unusual carbon sources such as hydrocarbons, its potential as a host for expression of heterologous proteins and its ability to shift between a yeast and a hyphal form (for reviews, see references 3 and 4). Surprisingly not much information is available on the enzymes that participate in the main catabolic routes (22). Since Y. lipolytica is an obligately respiratory organism, catabolism of all carbon sources transits through the tricarboxylic acid cycle. This is a significant difference with fermentative yeasts such as Saccharomyces cerevisiae, in which catabolism of glucose and other sugars proceeds under many conditions, mainly by fermentation.The tricarboxylic acid cycle has an amphibolic role in metabolism; it functions not only as an oxidative device coupled to energy production but also provides building blocks for the synthesis of important molecules such as porphyrins and several amino acids (Fig. 1). Withdrawal of the intermediates of the cycle for this last purpose would cause a stop of its function if there were no other reactions to replenish it. In yeasts and fungi growing in minimal medium, the two known ways to replenish the tricarboxylic acid cycle are the reaction catalyzed by pyruvate carboxylase and those catalyzed by isocitrate lyase and malate synthase, which form the glyoxylate bypass (Fig. 1). Pyruvate carboxylase coupled with phosphoenolpyruvate-carboxykinase also serves during growth in some nonsugar substrates to provide phosphoenolpyruvate since the pyruvate kinase reaction is irreversible under physiologica...

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“…Under conditions of glucose excess, the activity of pyruvate kinase should be high enough to provide sufficient amounts of ATP and pyruvate, which is a central metabolite involved in a variety of metabolic pathways. On the other hand, under gluconeogenic conditions, down-regulation of the Pyk1p-catalyzed reaction is necessary in order to avoid a futile cycling between pyruvate and PEP via the ATP-consuming reactions catalyzed by pyruvate carboxylase and PEP carboxykinase (15,51,53). Accordingly, the concentration of FBP is high in cells growing on fermentable carbon sources.…”

mentioning

confidence: 99%

Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate

et al. 1997

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We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.Pyruvate kinase is the last enzyme in the glycolytic pathway of sugar catabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate (PEP) into pyruvate by the addition of a proton and the loss of a phosphate group, which is transferred to ADP. Pyruvate kinases from a wide range of organisms have been extensively studied, and much is known about their physical and catalytic properties (8,35,38,39). Nearly all characterized eukaryotic pyruvate kinases are tightly regulated and are activated by fructose-1,6-bisphosphate (FBP). The mammalian muscle isoenzyme M1 is the only known pyruvate kinase that displays hyperbolic kinetics and lacks allosteric control (40). On the other hand, pyruvate kinases from prokaryotes can be activated by either FBP or other sugar phosphates (e.g., ribose phosphate), and those from trypanosomes can be activated by fructose-2,6-bisphosphate (35).In the yeast Saccharomyces cerevisiae, pyruvate kinase has been thought to be encoded solely by the PYK1 gene. The gene was cloned and sequenced by Burke et al. (16), and part of the nucleotide sequence was revised by McNally et al. (36). PYK1 codes for a 54.5-kDa protein (Pyk1p) consisting of 500 amino acids. Mutants defective in the PYK1 gene fail to grow on fermentable carbon sources and are even inhibited by them (17,18,30). However, they grow normally on ethanol or other gluconeogenic carbon sources. Under those conditions, hexose phosphates are provided by the gluconeogenic pathway, which uses the enzymes FBP and PEP carboxykinase to bypass the 6-phosphofructo-1-kinase and pyruvate kinase reactions, respectively. The concentrations of glycolytic metabolites in pyk1 deletion mutants after growth on an ethanol-containing medium are similar to the wild-type levels, but after addition of glucose, a large increase in the amounts of PEP and phosphoglycerates can be observed (12, 17). These results suggested that Pyk1p is the main enzyme which catalyzes the conversion of PEP into pyruvate in S. cerevisiae.The biochemical properties of yeast Pyk1p suggest that it plays a central regul...

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Regulation of Pyruvate Carboxylase Isozyme (PYC1, PYC2) Gene Expression in Saccharomyces cerevisiae during Fermentative and Nonfermentative Growth

Cited by 78 publications

References 0 publications

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae

Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype

Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate

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