In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ∼16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria.
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Research Article|
December 21 2006
Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism
Paul G. Crichton;
Paul G. Crichton
1
1Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
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Charles Affourtit;
Charles Affourtit
1
1Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
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Anthony L. Moore
Anthony L. Moore
2
1Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
2To whom correspondence should be addressed (email a.l.moore@sussex.ac.uk).
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Publisher: Portland Press Ltd
Received:
August 02 2006
Revision Received:
September 13 2006
Accepted:
September 25 2006
Accepted Manuscript online:
September 25 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2007
Biochem J (2007) 401 (2): 459–464.
Article history
Received:
August 02 2006
Revision Received:
September 13 2006
Accepted:
September 25 2006
Accepted Manuscript online:
September 25 2006
Citation
Paul G. Crichton, Charles Affourtit, Anthony L. Moore; Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism. Biochem J 15 January 2007; 401 (2): 459–464. doi: https://doi.org/10.1042/BJ20061181
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