Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae.
Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.
Ter Schure
Eelko G
EG
1Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Silljé
Herman H W
HHW
1Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Raeven
Leon J R M
LJRM
1Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Boonstra
Johannes
J
1Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Verkleij
Arie J
AJ
1Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Verrips
C Theo
CT
2Unilever Research Laboratorium Vlaardingen, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands.|1Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
D026905:Amino Acid Transport Systems; D000641:Ammonia / Q000378:metabolism; D000643:Ammonium Chloride / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
D026905:Amino Acid Transport Systems; D017931:DNA Primers; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D000643:Ammonium Chloride; D000641:Ammonia; D005969:Glutamate Dehydrogenase; D005974:Glutamate-Ammonia Ligase; D005947:Glucose; D009584:Nitrogen