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Eukaryotic Cell, August 2003, p. 809-820, Vol. 2, No. 4
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.4.809-820.2003

POS5 Gene of Saccharomyces cerevisiae Encodes a Mitochondrial NADH Kinase Required for Stability of Mitochondrial DNA

Micheline K. Strand,1,{dagger} Gregory R. Stuart,1,2 Matthew J. Longley,1 Maria A. Graziewicz,1 Olivia C. Dominick,1 and William C. Copeland1*

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709,1 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 277102

Received 1 May 2003/ Accepted 15 May 2003

In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD+, with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8m reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases.

* Corresponding author. Mailing address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, MD E3-01, Research Triangle Park, NC 27709. Phone: (919) 541-4792. Fax: (919) 541-7613. E-mail: copelan1{at}niehs.nih.gov.

{dagger} Present address: Army Research Office, Biosciences Division, Research Triangle Park, NC 27709.

Eukaryotic Cell, August 2003, p. 809-820, Vol. 2, No. 4
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.4.809-820.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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