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Journal of Bacteriology, May 2000, p. 2823-2830, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

In Vivo Analysis of the Mechanisms for Oxidation of Cytosolic NADH by Saccharomyces cerevisiae Mitochondria

Karin M. Overkamp,1 Barbara M. Bakker,1 Peter Kötter,2 Arjen van Tuijl,1 Simon de Vries,1 Johannes P. van Dijken,1 and Jack T. Pronk1,*

Kluyver Laboratory of Biotechnology, Delft University of Technology, NL-2628 BC Delft, The Netherlands,1 and Institut für Mikrobiologie, J. W. Goethe Universität Frankfurt, Biozentrum N250, 60439 Frankfurt, Germany2

Received 28 December 1999/Accepted 22 February 2000

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.

* Corresponding author. Mailing address: Kluyer Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands. Phone: 31 15 278 3214. Fax: 31 15 278 2355. E-mail: j.t.pronk{at}stm.tudelft.nl.

Journal of Bacteriology, May 2000, p. 2823-2830, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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