Abstract
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.
Highlights
NADP is required by the pentose phosphate pathway, and NADPH is required for resistance to oxidative stresses
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]
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]
Summary
Enzyme Expression and Purification—Expression plasmids were designed for purification of His-tagged versions of Utr and Yef from Escherichia coli. E. coli BL21 cells transformed with either plasmid pB421 or pB430 were used to express the His-tagged NAD kinases. The UTR1 gene PCR product and plasmid pRS416 [35]. The POS5 gene PCR product and pRS416 were digested with BamHI and XhoI and ligated to produce pB532. BY274 heterozygous for pos, utr, and yef disruption was transformed with pB532, pB534, and pB535 and, after sporulation and tetrad dissection, haploids were isolated with each plasmid present in each combination of NAD/NADH kinase gene disruption. For POS5, primers 7343 and 7345 were used to amplify The NAD/NADH kinase reaction with ATP as phosphoryl the Geneticin resistance marker of plasmid pRS401 [35]. Because yeast NAD kinase is a relatively micromanipulation, and the genotypes of the resulting haploid unstable enzyme [44], our rapid, one-step purification pro-
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