Effects of a Mitochondrial Mutator Mutation in Yeast POS5 NADH Kinase on Mitochondrial Nucleotides

Linda J Wheeler,Christopher K Mathews

doi:10.1074/jbc.m112.394031

Linda J Wheeler, Christopher K Mathews

Open Access

https://doi.org/10.1074/jbc.m112.394031

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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.

Highlights

A mutation in the yeast gene POS5, which encodes mitochondrial NADH kinase, is a mitochondrial mutator
In previous investigations with rat tissue mitochondria, we identified aspects of DNA precursor metabolism that probably contribute to this difference: 1) a strikingly asymmetric distribution of deoxyribonucleoside triphosphates2 within the mitochondrion [3] and 2) the status of the elevated mitochondrial dGTP pool as a target
DNTP Pools—We expected the perturbation of mitochondrial pyridine nucleotide metabolism to affect the pool sizes of deoxyribonucleoside triphosphate (dNTP) within the mitochondrion because NADPH is involved directly in the dihydrofolate reductase-thymidylate synthase cycle and more indirectly in ribonucleotide reduction through reduction of oxidized glutaredoxin or thioredoxin

Summary

Introduction

A mutation in the yeast gene POS5, which encodes mitochondrial NADH kinase, is a mitochondrial mutator. An informative way to determine whether the POS5 mutation affects mutagenesis through dNTP metabolic abnormalities is to measure dNTP pools directly in extracts of isolated yeast mitochondria. When we analyzed yeast mitochondrial extracts, we did see an effect of the pos5 mutation on dNTP pools.

Results

Conclusion