In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, b ecause the mitochondrial inner membrane is impermeable to NADH. In Saccharo myces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or N de2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. Thi s study addresses the physiological relevance of these mechanisms and the p ossible involvement of alternative routes for mitochondrial oxidation of cy tosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2 Delta m utant exhibited fully respiratory growth at low specific growth rates. Alco holic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h(-1)). Apparently, the glycerol-3-phosphate shuttle is not e ssential for respiratory glucose dissimilation. An nde1 Delta nde2 Delta mu tant already produced glycerol at specific growth rates of 0.10 h(-1) and a bove, indicating a requirement for external NADH dehydrogenase to sustain f ully respiratory growth. An nde1 Delta nde2 Delta gut2 Delta mutant produce d even larger amounts of glycerol at specific growth rates ranging from 0.0 5 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mec hanisms could not fully replace the external NADH dehydrogenases and glycer ol-3-phosphate shuttle. However, at low dilution rates, the nde1 Delta nde2 Delta gut2 Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system h as to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intrami tochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxida tion to the internal NADH dehydrogenase.