POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA.
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 arg8(m) 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.
Strand
Micheline K
MK
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Stuart
Gregory R
GR
Longley
Matthew J
MJ
Graziewicz
Maria A
MA
Dominick
Olivia C
OC
Copeland
William C
WC
D000255:Adenosine Triphosphate / Q000378:metabolism; D002478:Cells, Cultured; D003300:Copper / Q000494:pharmacology; D004272:DNA, Mitochondrial / Q000235:genetics*; D004734:Energy Metabolism / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics*; D028361:Mitochondrial Diseases / Q000201:enzymology / Q000235:genetics; D024101:Mitochondrial Proteins; D009154:Mutation / Q000235:genetics; D009243:NAD; D018384:Oxidative Stress / Q000235:genetics; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*
D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D003300:Copper; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C060670:POS5 protein, S cerevisiae