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

Abstract

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.

Synthetic Lethal and Biochemical Analyses of NAD and NADH Kinases in Saccharomyces cerevisiae Establish Separation of Cellular Functions

“…4B). Since NADPH is used as a cofactor by enzymes involved in the detoxification of peroxide, superoxide, and hydroxyl radicals that are generated in mitochondria (48 -50), depletion of the mitochondrial NADPH pool would be expected to lead to an increased level of oxidative damage to mitochondrial proteins and DNA, consistent with the mitochondrial genome instability that results from pos5 mutation (2). In contrast to the respiratory incompetence of pos5 mutants, utr1 mutants displayed wild-type growth on glycerol (Fig.…”

“…Interestingly, transforming freshly prepared pos5 mutants with a POS5 plasmid restores resistance to reactive oxygen species and growth on nonfermentable carbon sources, but older pos5 mutants lose the ability to have their respiratory defect restored by Pos5 expression. These data suggest that mitochondrial NADPH-dependent antioxidant reductases are required to protect mitochondria from reactive oxygen species damage and that this damage can lead to irreparable lesions to the mitochondrial genome (1,2). In the cytosol, NADP is used by pentose phosphate pathway enzymes glucose-6-phosphate dehydrogenase Zwf1 and 6-phosphogluconate dehydrogenase Gnd1 and -2 to produce ribulose-5-phosphate and NADPH (3).…”

“…Deletion mutants in the gene encoding mitochondrial matrix NADH kinase, POS5, are sensitive to growth in hyperoxia, H 2 O 2 , and methyl viologen and are also respiratory-deficient (1,2). Interestingly, transforming freshly prepared pos5 mutants with a POS5 plasmid restores resistance to reactive oxygen species and growth on nonfermentable carbon sources, but older pos5 mutants lose the ability to have their respiratory defect restored by Pos5 expression.…”

“…Phylogenetic analysis suggests that the former enzyme, nicotinamidase, found in fungi (23), and the latter enzyme, nicotinamide phosphoribosyltransferase, found in vertebrates (24), entered eukaryotic lineages via horizontal gene transfer. 2 The final steps in eukaryotic NAD synthesis are catalyzed either by glutamine-dependent NAD synthetase (26) for the de novo nicotinic acid import and nicotinic acid salvage pathways that go through nicotinic acid mononucleotide (NaMN) or by nicotinic acid mononuleotide/NMN adenylyltransferase (27) for the nicotinamide riboside kinase and nicotinamide phosphoribosyltransferase pathways that go through an NMN intermediate. In the nucleus and cytosol, NADdependent hydride transfer enzymes such as inosine monophosphate dehydrogenase, alcohol dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase reduce NAD to NADH.…”

Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress)

“…Binary sequence alignments of NADK3 revealed negligible similarity to either of the previously identified Arabidopsis NADKs. However, sequence analysis revealed that NADK3 possessed similarity to POS5 (YPL 188W) from yeast (S. cerevisiae), recently shown to be an NADHK [13,14]. This enzyme, which to date has only been described in yeast, is distinct from NADK [15,16] and localizes to the mitochondria [13,14,17].…”