The Saccharomyces cerevisiae NDE1 andNDE2 Genes Encode Separate Mitochondrial NADH Dehydrogenases Catalyzing the Oxidation of Cytosolic NADH

doi:10.1074/jbc.273.38.24529

The Saccharomyces cerevisiae NDE1 andNDE2 Genes Encode Separate Mitochondrial NADH Dehydrogenases Catalyzing the Oxidation of Cytosolic NADH*

From the ^‡Department of Microbiology and Enzymology, Kluyver Institute of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands, and ^§Institut für Mikrobiologie, Goethe Universität Frankfurt, Marie-Curie Strasse 9, Biozentrum N250, 60439 Frankfurt, Germany

Abstract

In Saccharomyces cerevisiae, theNDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane. In addition to this NADH dehydrogenase,S. cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane. To investigate whether open reading framesYMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter enzyme, NADH-dependent mitochondrial respiration was assayed in wild-type S. cerevisiae and nde deletion mutants. Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0.10 h⁻¹, in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration. Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in annde1Δ mutant and unaffected in an nde2Δmutant. NADH-dependent mitochondrial respiration was completely abolished in an nde1Δ nde2Δ double mutant. Mitochondrial respiration of substrates other than NADH was not affected in nde mutants. In shake flasks, an nde1Δ nde2Δ mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose. Glucose metabolism in aerobic, glucose-limited chemostat cultures (D = 0.10 h⁻¹) of an nde1Δ nde2Δ mutant was essentially respiratory. Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S. cerevisiae.

Footnotes

↵* This work is part of the Delft University DIOC-6 program “Mastering the Molecules of Manufacturing” and of the project “From Gene to Product in Yeast, a Quantitative Approach,” which is subsidized by the European Community (EC Framework IV Cell Factory Program).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed. Tel.: 31 15 278 3214; Fax: 31 15 278 2355; E-mail: j.t.pronk{at}stm.tudelft.nl.
↵2 S. de Vries, unpublished data.
Abbreviations:

SFH

short flanking homology

PCR

polymerase chain reaction.
- Received May 13, 1998.
- Revision received July 17, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.