DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains

Abstract

A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.

Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae

“…Therefore, the effects of nitrogen sources on each of the two isoenzymes could be examined using strains bearing a deletion in PYC1 or PYC2 genes, owing to the fact that the growth behaviour is not impaired in the respective pyc mutants. As previously reported [4,22], mutants in only one of the PYC genes or the double pyc1pyc2 mutant grew as the wild type in a glucose medium containing aspartate as the nitrogen source [4,22]. However, loss of PYC1 function resulted in a twofold reduction in the growth rate in a glucose/ammonium medium ( Table 2), suggesting that the function of this gene in anabolic pathways (i.e.…”

“…The physiological significance of the existence of two genes encoding pyruvate carboxylase in S. cerevisiae without any striking differences in their expression patterns and without known differences in the properties of their encoded enzymes has been a puzzle [4,5,7]. We have shown in this work, both by enzymatic assays and by Northern analysis, that there is an important difference in the response of PYC1 and PYC2 to the nitrogen source of the medium with independence of the carbon source.…”

“…Strain JF1209 (Mat a MAL2±8C SUC2 Dpyc1::KanMX4 Dpyc2::ILV2 SMR ) was obtained after a cross with CENPK113± 1 A and CEN.JB4 followed by a second cross between a pyc2 mutant segregant from the first cross with CEN.JB8. The pyc1pyc2 double mutant was identified by its inability to growth in a glucose mineral medium in the absence of aspartate [4]. Transformation of strain CEN.PK113±5D with plasmid pJC1 or pJC2±2 carrying a PYC1-lacZ or PYC2-lacZ gene fusion, respectively [7] was made according to [13].…”

“…The RNAs were separated in formaldehyde±agarose gels as described previously [16] and transferred to Hybond N 1 filters (Amersham, UK). Probes (the 1.1-kbp Pst I internal of PYC1 from pCG50 [4] and a 1.5-kb internal fragment of the ACT1 gene) were used for hybridization as recommended by the manufacturer. Blots were probed with 32 P-radiolabelled fragments of PYC1 and ACT1 using the megaprime TM DNA labelling system (Amersham, UK).…”

“…This key function justifies the fine metabolic control of this enzyme by different effectors, including activators such as acetyl-CoA and palmitoyl-CoA and inhibitors such as aspartate [1±3]. Saccharomyces cerevisiae contains two different genes, PYC1 and PYC2, encoding isoenzymes I and II of pyruvate carboxylase [4,5]. The physiological significance of the two genes is not yet clear, because even if both genes are expressed slightly differently depending on the growth phase of yeast cultures on glucose or on the carbon source of the medium, the differences appear minor as to physiological relevance [6,7].…”