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, glucoselimited chemostat cultures of a gut2⌬ 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 nde1⌬ nde2⌬ 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 nde1⌬ nde2⌬ gut2⌬ 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 nde1⌬ nde2⌬ gut2⌬ 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.As in other eukaryotes, respiratory dissimilation of sugars by Saccharomyces cerevisiae leads to the reduction of NAD ϩ to NADH in separate cellular compartments. Cytosolic NADH is produced by the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, as well as in assimilatory reactions (1, 30). In the mitochondrial matrix, NADH is formed by the tricarboxylic acid cycle and the pyruvate-dehydrogenase complex. Under anaerobic conditions, glucose dissimilation occurs exclusively via alcoholic fermentation, which is a redox-neutral process. The additional NADH originating from biomass production can be reoxidized via glycerol production (30). Under aerobic conditions, glycerol production is not necessary, because the reoxidation of cytosolic NADH can be coupled to the mitochondrial respiratory chain.Although the outer mitochondrial membrane is permeable to NADH (17), the inner membrane is not (35). Therefore, coupling of NADH reoxidation to the respiratory chain has to occur on both sides of the mitochondrial inner membrane. In plant mitochondria, cytosolic NADH can be oxidized either by an external NADH dehydrogenase or by a redox shuttle (14), whereas in the mitochondrial mat...

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Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture

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