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...

Saccharomyces cerevisiae HTR1 mutants are severely impaired in the uptake of glucose. We have cloned dominant HTR1 mutant alleles and show that they encode mutant forms of the Mth1 protein. Mth1 is shown to be involved in carbon source-dependent regulation of its own, invertase and hexose transporter gene expression. The mutant forms block the transduction of the Snf3-and Rgt2-mediated glucose signals upstream of the Rgt1 transcriptional regulator.HTR1 mutants show severely reduced glucose uptake rates (12). They were obtained by selecting for revertants of triosephosphate isomerase (tpi1⌬) mutants of Saccharomyces cerevisiae which had overcome the strong inhibitory effect of glucose on growth and carbon metabolism (12). The transcription of various known glucose transporter genes (HXT1, HXT3, and HXT4) is defective in HTR1 mutants. It was speculated that mutations in HTR1 affect a negative factor of hexose transporter gene expression.(This work is part of the Ph.D. theses of F. Schulte and R. Wieczorke from Heinrich-Heine-Universität, Düsseldorf, Germany.)To identify the dominant HTR1 allele, a DNA library was constructed from the HTR1-23 mutant strain (12). Genomic DNA was partially digested with Sau3A and cloned into the BamHI-digested vector YCp50. This library was transformed into the yeast wild-type strain MC971A (MATa ura3-52 his3-11,15 MAL2 SUC2 GAL MEL) and plated onto YNB medium (0.67% yeast nitrogen base, supplemented for auxotrophic demands) lacking uracil with 2% glucose. A total of about 10,000 transformants were replica plated onto YNB medium lacking uracil with 2% galactose additionally supplemented with 100-ppm 2-deoxyglucose (2-DOG), a toxic analogue of glucose (1), because HTR1 mutants are resistant to 2-DOG. The plasmids of two transformants which were able to grow in the presence of 2-DOG were isolated, amplified in Escherichia coli DH5␣FЈ, and retransformed into yeast strain MC971A. One of the plasmids (pHTR1-23) conferred growth in the presence of 2-DOG to all of the transformants. A 5.9-kb HindIII/SalI fragment containing the complete insertion of plasmid pHTR1-23 was recloned into the integrative vector YIp5. This plasmid was linearized with XhoI within the inserted fragment and integrated into the genome of the original HTR1-23 mutant strain. The correct integration into the homologous genomic region was confirmed by Southern analysis. The strain was crossed with the isogenic wild-type strain MC996A 112 MAL2 SUC2 GAL MEL) and a tetrad analysis was performed. Eighteen tetrads were all parental ditype with regard to resistance to 2-DOG and uracil prototrophy, indicating that the cloned fragment is tightly linked to the dominant HTR1-23 allele.The dominant HTR1-23 gene was localized on a 2.6-kb EcoRI/(Sau3A-BamHI) fragment, which contains the complete MTH1 gene. This gene originally had been isolated as a homologue of the STD1 (also known as MSN3) gene (4, 7). Both proteins have been shown to modulate glucose-regulated expression of SUC2 and HXT1-4 (7, 18, 19). The DNA sequence of the ent...

Glucose repression in the yeast Saccharomyces cerevisiae designates a global regulatory system controlling the expression of various sets of genes required for the utilization of alternate carbon sources. In a screen, designed for the selection of mutants with reduced glycolytic flux we obtained isolates which were shown by complementation of the cloned wild-type gene to be allelic to the glucose repression mutants grrllcat80lcot2 previously described. We demonstrate that the grrl lesion lead to a concentration-dependent decrease in glycolytic flux on glucose. It is very likely that this is caused by a significant decrease in the expression of various genes encoding hexose transporters (HXTI,3) leading to a reduced glucose-uptake rate. In contrast, expression of the maltose permease gene (MALll) and maltose utilization is normal. There is indirect evidence that grrl affects the uptake of amino acids, and others have shown that the sugar-induced transport of divalent cations is impaired. These effects are not glucose-specific. We suggest that Grrl, a putative cytoplasmic protein, has a central function in the sensing of nutritional conditions for a variety of unrelated substances, and that relief from glucose repression may be a corollary of this defect in sensing.In the yeast Saccharomyces cerevisiae glucose or other readily fermentable carbon sources act as global regulators of growth and metabolism. These regulatory effects are executed partly at the transcriptional level. Glucose repression and glucose induction designate mechanisms that exert specific control on glucose-sensitive genes. Glucose-repressible genes include SUC2 (invertase) and genes that are required for the utilization of alternate carbohydrates (the GAL, MAL and MEL regulons required for the utilization of galactose, maltose and melibiose, respectively; for review see [l]), mitochondrial genes [2] and nuclear genes for mitochondrial and gluconeogenic functions [3]. Analysis of glucose induction at the gene expression level is less advanced. Nevertheless, glucose or its metabolism exerts a plethora of activating effects on many cellular functions. It is not clear, in which way glucose repression and induction are functionally linked.Glucose repression mechanisms in S. cerevisiae have been revealed by means of appropriate mutants either defective in repression or in derepression (for review, see [4]). Analysis of individual mutants allowed in some cases the identification or localization of the regulatory defect. A number of such mutants lead to the identification of transcription factors or transcriptional regulators of glucose-sensitive genes. There are a number of mutants which apparently affect functions early in the signalling pathway for glucoseCorrespondence to