Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, the existence of PYC1 and PYC2 encoding cytosolic pyruvate carboxylase isoform I and II is rather puzzling, owing to the lack of potent differential gene regulation by the carbon sources. We report several findings indicating that these two genes are differentially regulated by the nature of the nitrogen source. In wild-type cells, the activity of pyruvate carboxylase, which is the sum of pyruvate carboxylase isoform I and II, was two- to fivefold lower in carbon medium containing aspartate, asparagine, glutamate or glutamine instead of ammonium as the nitrogen source, whereas it was 1.5- to threefold higher when the ammonium source was substituted by arginine, methionine, threonine or leucine. These enzymatic changes were independent of the nature of the carbon source and closely correlated to the changes in beta-galactosidase from PYC1-lacZ gene fusion and in PYC1 transcripts. Transfer of exponentially growing cells of the pyc2 mutant from an aspartate or a glutamate medium to an ammonium medium caused a fivefold increase in PYC1 mRNA in less than 30 min, whereas in the inverse experiment, PYC1 transcripts returned within 30 min to the low levels found in aspartate/glutamate medium. By contrast, these conditions affected neither the pyruvate carboxylase activity encoded by PYC2 nor PYC2 mRNA. Considering that changes in PYC1 expression inversely correlated with changes in alpha-ketoglutarate concentration or in alpha-ketoglutarate/glutamate ratio following the nitrogen shift experiments, and taking into account the pivotal role of this metabolite in ammonium assimilation, it is suggested that changes in alpha-ketoglutarate or in the alpha-ketoglutarate/glutamate ratio might be implicated in triggering the nitrogen effects on PYC1 expression. The physiological significance of the differential sensitivity of PYC1 and PYC2 genes with respect to the nitrogen source in the growth medium is also discussed.
Huet
C
C
Centre de Bioingénierie Gilbert Durand, Toulouse,France.
Menendez
J
J
Gancedo
C
C
François
J M
JM
D000596:Amino Acids / Q000737:chemistry; D015152:Blotting, Northern; D002244:Carbon / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D005947:Glucose / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007763:Lac Operon; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism*; D020033:Protein Isoforms; D011766:Pyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D000596:Amino Acids; D007656:Ketoglutaric Acids; D020033:Protein Isoforms; D012333:RNA, Messenger; D012313:RNA; D002244:Carbon; D001616:beta-Galactosidase; D011766:Pyruvate Carboxylase; D005947:Glucose; D009584:Nitrogen