Effects of a mitochondrial mutator mutation in yeast POS5 NADH kinase on mitochondrial nucleotides

Abstract

Saccharomyces cerevisiae contains three NADH/NAD(+) kinases, one of which is localized in mitochondria and phosphorylates NADH in preference to NAD(+). Strand et al. reported that a yeast mutation in POS5, which encodes the mitochondrial NADH kinase, is a mutator, specific for mitochondrial genes (Strand, M. K., Stuart, G. R., Longley, M. J., Graziewicz, M. A., Dominick, O. C., and Copeland, W. C. (2003) Eukaryot. Cell 2, 809-820). Because of the involvement of NADPH in deoxyribonucleotide biosynthesis, we asked whether mitochondria in a pos5 deletion mutant contain abnormal deoxyribonucleoside triphosphate (dNTP) pools. We found the pools of the four dNTPs to be more than doubled in mutant mitochondrial extracts relative to wild-type mitochondrial extracts. This might partly explain the mitochondrial mutator phenotype. However, the loss of antioxidant protection is also likely to be significant. To this end, we measured pyridine nucleotide pools in mutant and wild-type mitochondrial extracts and found NADPH levels to be diminished by ∼4-fold in Δpos5 mitochondrial extracts, with NADP(+) diminished to a lesser degree. Our data suggest that both dNTP abnormalities and lack of antioxidant protection contribute to elevated mitochondrial gene mutagenesis in cells lacking the mitochondrial NADH kinase. The data also confirm previous reports of the specific function of Pos5p in mitochondrial NADP(+) and NADPH biosynthesis.

FIGURE 1.

Yeast mitochondrial dNTP pools. Data represent means ± S.D. from six independent experiments.

FIGURE 2.

Yeast cytosolic dNTP pools. Data represent means ± S.D. from six independent experiments.

FIGURE 3.

Substrate saturation curves for a hypothetical but realistic DNA polymerase with K_m values of 1 μm for chain extension from a properly base-paired 3′ terminus and 1000 μm for extension from a mismatched 3′ terminus. Kinetic data for extension from properly and improperly matched termini are compiled in Ref. .

SCHEME 1

FIGURE 4.

Yeast mitochondrial pyridine nucleotide pools. Data represent means ± S.D. from five independent experiments.

FIGURE 5.

Yeast cytosolic NADPH and NADP⁺ pools. Data represent means ± S.D. from four independent experiments.