The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant.
Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase. Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.
Boles
E
E
Institut für Mikrobiologie, Technische Hochschule Darmstadt, Germany.
Lehnert
W
W
Zimmermann
F K
FK
D000595:Amino Acid Sequence; D001483:Base Sequence; D017353:Gene Deletion; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism*; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics* / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007656:Ketoglutaric Acids / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism / Q000494:pharmacology*; D009249:NADP / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D013489:Suppression, Genetic; D014170:Transformation, Genetic
D005971:Glutamates; D007656:Ketoglutaric Acids; D009243:NAD; D018698:Glutamic Acid; D009249:NADP; D005969:Glutamate Dehydrogenase; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose