Multiple copies of the pyruvate kinase gene affect yeast cell growth

The Saccharomyces cerevisiae pyruvate kinase gene (PYK1) was transformed into yeast using the multicopy vector pJDB207. Growth rates and PYK1 gene expression levels varied considerably amongst the transformants. Yeast transformants expressing the PYK1 gene at high levels formed small colonies compared with those expressing the gene at relatively low levels. Slow-growing transformants 'reverted' at high frequency to more rapid growth, and this correlated with decreases in PYK1 gene copy number and PYK1 mRNA abundance. This apparent selection against PYK1 over-expression was disrupted by the introduction of a stop codon at the 5'-end of the PYK1 coding region, thus confirming that the growth effects were mediated by the PYK1 gene. However, massive overproduction of pyruvate kinase in yeast, using multiple copies of a PGK:PYK gene fusion, had no significant effect upon cell growth. This suggests that the deleterious effect upon the host yeast cell is mediated by abnormally high levels of the wild-type gene or PYK1 mRNA, rather than by increased pyruvate kinase levels.     

Regulation of fitness in yeast overexpressing glycolytic enzymes: parameters of growth and viability.

Current models predict that large increases over wild-type in the activity of one enzyme will not alter an organism's fitness. This prediction is tested in Saccharomyces cerevisiae through the use of a high copy plasmid that bears one of the following: hexokinase B (HEXB), phosphoglucose isomerase (PGI), phosphofructokinase (PFKA and PFKB), or pyruvate kinase (PYK). Transformants containing these plasmids demonstrate a four to ten-fold increase in enzyme specific activity over either the parent strain or transformants containing the plasmid alone. Haploid and diploid transformants derived from independent backgrounds were grown on both fermentable and non-fermentable carbon sources and evaluated for several components of fitness. These include growth rate under non-limiting conditions, maximum stationary phase density, and viability in extended batch culture. Cell viability is not affected by overproduction of these enzymes. Growth rate and stationary phase density do not differ significantly among strains that overexpress HEXB, PGI or contain the vector alone. PFKA, B transformants show reduced growth rate on glucose in one background only. For these loci the current model is confirmed. By contrast, when grown on glucose, yeast overexpressing PYK demonstrate reduced growth rate and increased stationary phase density in both backgrounds. These effects are abolished in cells containing plasmids with a Tn5 disrupted copy of the PYK gene. Our results are consistent with reports that the PYK locus may exert control over the yeast cell cycle and suggest that it will be challenging to model relations between fitness and activity for multifunctional proteins.     

Regulation of fitness in yeast overexpressing glycolytic enzymes: responses to heat shock and nitrogen starvation.

Current models based on the analysis of linear metabolic pathways at steady-state predict that large increases over wild type in the activity of one enzyme will not alter an organism's fitness. This prediction is tested at steps in a highly branched pathway under two conditions known to alter steady-state: heat shock and nitrogen starvation. Saccharomyces cerevisiae transformants overproducing 1 of 4 enzymes in glycolysis (hexokinase B, phosphoglucose isomerase, phosphofructokinase, or pyruvate kinase) were subjected to heat shock in both exponential and stationary phases of growth. In neither phase does enzyme overexpression alter heat shock sensitivity. When starved for nitrogen in acetate medium, transformants overproducing hexokinase, phosphoglucose isomerase, and phosphofructokinase sporulate at the same rate and with the same frequency as cells harbouring only the plasmid vector. Current models therefore correctly predict the relationship between activity and components of fitness for 3 of 4 enzymes. By contrast, cells overexpressing pyruvate kinase sporulate poorly. This defect is not observed among cells transformed with a plasmid containing a Tn5 disrupted copy of the PYK gene. These findings are consistent with reports that implicate the PYK locus in yeast cell cycle control and suggest that it may be challenging to model relations between fitness and activity for multifunctional proteins.     

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

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.     

Leishmania donovani: characterization and expression of ORFF, a gene amplified from the LDI locus

The LD1 locus is a 27.5-kb region of chromosome 35 that is conserved among all species of Leishmania and is amplified in several different isolates. Here, we report the genomic distribution of ORFF, a gene from the LD1 region, and its expression at the RNA and protein levels in two Indian isolates of Leishmania donovani. In both of these isolates, ORFF was present as a single copy on chromosome 35. Densitometric analysis of ORFF mRNA abundance revealed relative abundance of 0.2 and 1.0 in AG83 and S-Lal, respectively. Antiserum against recombinant ORFF protein detected a protein of the predicted size ( approximately 34 kDa) in both strains. The protein is most abundant in mid-log-phase promastigotes and has a nuclear localization. The ORFF protein is preferentially expressed in L. donovani amastigotes but, in contrast, is expressed at higher levels in L. major promastigotes.     

G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription

G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.     

The isolation and characterization of the pyruvate kinase-encoding gene from the yeast Yarrowia lipolytica.

The dimorphic yeast, Yarrowia lipolytica, has been developed as a useful expression/secretion system for heterologous proteins such as chymosin and tissue plasminogen activator. To further develop this expression system, we have cloned the gene (PYK) encoding the highly expressed glycolytic enzyme, pyruvate kinase (PYK). Genomic clones were selected by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that were conserved through evolution. The clones identified by hybridization contained overlapping DNA inserts. We have confirmed the identity of the cloned gene based on two criteria: (1) the nucleotide sequence of the proposed PYK gene predicts a protein that is highly homologous to the corresponding Saccharomyces cerevisiae enzyme, and (2) PYK-specific activity was increased twofold when wild-type Y. lipolytica strains were transformed with the isolated DNA. Interestingly, we found that the open reading frame of the Y. lipolytica PYK gene was interrupted by an intron. This represents the first report of an intron in a Y. lipolytica gene.