Analysis of transcription and translation of glycolytic enzymes in glucose-limited continuous cultures of Saccharomyces cerevisiae.
mRNA steady-state levels and activities of enzymes of intermediary carbon metabolism (hexokinase, phosphoglucoisomerase, phosphofructokinase, glucose-6-phosphate dehydrogenase, phosphoglucomutase) and glucose-regulated enzymes (pyruvate decarboxylase, pyruvate dehydrogenase, invertase, alcohol dehydrogenase) were determined in glucose-limited continuous cultures of an industrial strain of Saccharomyces cerevisiae at different dilution rates (D) ranging from 0.05 to 0.315 h-1. The activity of most enzymes measured remained constant over this range except for alcohol dehydrogenase I/II which decreased proportionally with increasing dilution rate. A decrease in phosphoglucomutase activity occurred with increasing dilution rate but reached a minimum at D 0.2 h-1 and from thereon remained constant. A decrease in pyruvate decarboxylase activity and a slight decrease in phosphoglucoisomerase activity was observed. At D 0.29/0.315 h-1, at the onset of the Crabtree effect, most glycolytic enzymes remained constant except for pyruvate decarboxylase and glucose-6-phosphate dehydrogenase which increased at D 0.315 h-1 and alcohol dehydrogenase I/II which decreased. The ADHI/II and PDC1 mRNA levels obtained at the different dilution rates were in accordance with the activity measurements. The mRNA level of HXK1 decreased with increasing dilution rates, whereas the transcription of HXK2 increased. Pyruvate dehydrogenase (PDA1) and PGI1 mRNA fluctuated but no significant change could be detected. These results indicate that there is no transcriptional or translational regulation of glycolytic flux between D 0.05 h-1 and 0.315 h-1 except at the branch point between oxidative and fermentative metabolism (pyruvate decarboxylase/pyruvate dehydrogenase) at D 0.315 h-1. Surprisingly regulation of the Crabtree effect does not seem to involve transcriptional regulation of PDA1.(ABSTRACT TRUNCATED AT 250 WORDS)
Sierkstra
L N
LN
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Verbakel
J M
JM
Verrips
C T
CT
D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism; D002455:Cell Division; D004794:Enzyme Repression; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010101:Oxygen Consumption; D014176:Protein Biosynthesis / Q000187:drug effects; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D013403:Sugar Phosphates / Q000032:analysis; D014158:Transcription, Genetic / Q000187:drug effects
D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012333:RNA, Messenger; D013403:Sugar Phosphates; D002245:Carbon Dioxide; D005947:Glucose