Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria.
Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae. In S. cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactis rag2 null mutants still grow on glucose. It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K. lactis mitochondria to oxidize cytosolic NADPH. This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway. Consistent with this hypothesis, mitochondria of S. cerevisiae cannot oxidize NADPH. In the present study, the ability of K. lactis mitochondria to oxidize cytosolic NADPH was experimentally investigated. Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K. lactis strain CBS 2359 and from an isogenic rag2Delta strain. Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis. This activity was ca. 2.5-fold higher in the rag2Delta mutant than in the wild-type strain. In contrast to mitochondria from wild-type K. lactis, mitochondria from the rag2Delta mutant exhibited high rates of ethanol-dependent oxygen uptake. Subcellular fractionation studies demonstrated that, in the rag2Delta mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent 'acetaldehyde reductase' was also increased. These observations indicate that two mechanisms may participate in mitochondrial oxidation of cytosolic NADPH by K. lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP(+)- and NAD(+)-dependent alcohol dehydrogenases.
Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands. j.t.pronk@tudelft.nl
Karin M
KM
Overkamp
Barbara M
BM
Bakker
H Y
HY
Steensma
Johannes P
JP
van Dijken
Jack T
JT
Pronk
D002458:Cell Fractionation; D003600:Cytosol / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption
D009249:NADP; D009252:NADPH Dehydrogenase; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose