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J. Biol. Chem., Vol. 281, Issue 32, 22439-22445, August 11, 2006

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

Pawel Bieganowski, Heather F. Seidle, Marzena Wojcik^¶, and Charles Brenner¹

From the Departments of Genetics and Biochemistry and Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire 03756, International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street 02-109 Warsaw, Poland, and ^¶Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland

Production of NADP and NADPH depends on activity of NAD and NADH kinases. Here we characterized all combinations of mutants in yeast NAD and NADH kinases to determine their physiological roles. We constructed a diploid strain heterozygous for disruption of POS5, encoding mitochondrial NADH kinase, UTR1, cytosolic NAD kinase, and YEF1, a UTR1-homologous gene we characterized as encoding a low specific activity cytosolic NAD kinase. pos5 utr1 is a synthetic lethal combination rescued by plasmid-borne copies of the POS5 or UTR1 genes or by YEF1 driven by the ADH1 promoter. Respiratory-deficient and oxidative damage-sensitive defects in pos5 mutants were not made more deleterious by yef1 deletion, and a quantitative growth phenotype of pos5 and its arginine auxotrophy were repaired by plasmid-borne POS5 but not UTR1 or ADH1-driven YEF1. utr1 haploids have a slow growth phenotype on glucose not exacerbated by yef1 deletion but reversed by either plasmid-borne UTR1 or ADH1-driven YEF1. The defect in fermentative growth of utr1 mutants renders POS5 but not POS5-dependent mitochondrial genome maintenance essential because rho–utr1 derivatives are viable. Purified Yef1 has similar nucleoside triphosphate specificity but substantially lower specific activity and less discrimination in favor of NAD versus NADH phosphorylation than Utr1. Low expression and low intrinsic NAD kinase activity of Yef1 and the lack of phenotype associated with yef1 suggest that Utr1 and Pos5 are responsible for essentially all NAD/NADH kinase activity in vivo. The data are compatible with a model in which there is no exchange of NADP, NADPH, or cytoplasmic NAD/NADH kinase between nucleocytoplasmic and mitochondrial compartments, but the cytoplasm is exposed to mitochondrial NAD/NADH kinase during the transit of the molecule.

Received for publication, December 30, 2005 , and in revised form, June 5, 2006.

^* This work was supported in part by Polish Ministry of Science Grant 2 P04A 05029 (to P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table showing oligonucleotides used in this study.

¹ To whom correspondence should be addressed. Tel.: 603-653-9922; Fax: 603-653-9923; E-mail: charles.brenner{at}dartmouth.edu.

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