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

Abstract

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2Delta mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic 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 essential for respiratory glucose dissimilation. An nde1Delta nde2Delta mutant already produced glycerol at specific growth rates of 0.10 h(-1) and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An nde1Delta nde2Delta gut2Delta mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the nde1Delta nde2Delta gut2Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.

FIG. 1

Overview of possible mechanisms of oxidation of cytosolic NADH by mitochondria of S. cerevisiae. Abbreviations: R[H₂], reduced metabolite; R, oxidized metabolite; Gpd, cytosolic glycerol-3-phosphate dehydrogenase; Gut2, membrane-bound mitochondrial glycerol-3-phosphate dehydrogenase; Nde, external NADH dehydrogenase; Ndi, internal NADH dehydrogenase; Q, ubiquinon pool; bc1, cytochrome bc₁ complex; cox, cytochrome c oxidase.

FIG. 2

Effects of dilution rate in an aerobic, glucose-limited chemostat culture on biomass yield (Y_XS) and specific production rate of ethanol and glycerol (respectively q_ethanol and q_glycerol) for wild-type S. cerevisiae CEN.PK113-7D (data taken from reference 31) (A), gut2Δ mutant CEN.PK225-2C (B), nde1Δ nde2Δ mutant CEN.PK167-2B (C), and nde1Δ nde2Δ gut2Δ mutant CEN.PK263-5D (D). Apparently filled-in symbols are a result of overlapping data points from independent chemostat cultures.

FIG. 3

Effects of dilution rate in an aerobic, glucose-limited chemostat culture on the specific production rate of CO₂ (qCO₂) and the specific consumption rate of O₂ (qO₂) for wild-type S. cerevisiae CEN.PK113-7D (data taken from reference 31) (A), gut2Δ mutant CEN.PK225-2C (B), nde1Δ nde2Δ mutant CEN.PK167-2B (C), and nde1Δ nde2Δ gut2Δ mutant CEN.PK263-5D (D). Apparently filled-in symbols are a result of overlapping data points from independent chemostat cultures.

FIG. 4

Schematic representation of proposed metabolic pathways for the oxidation of cytosolic NADH in an S. cerevisiae nde1Δ nde2Δ gut2Δ mutant, grown in an aerobic, glucose-limited chemostat culture at low dilution rates. (A) Conversion of dissimilatory glucose into equimolar amounts of pyruvate and glycerol; (B) consumption of ethanol produced in cytosol by mitochondria; (C) ethanol-acetaldehyde shuttle.