The Effect Of Mitochondrial NAD Kinase On Biological Oxidation Function In Saccharomyces Cerevisiae

NADPH is one of the most important co-enzymes in cells, not only for providing of the reducing equivalents in biosynthetic reactions, but also for protection of cells against the toxicity of ROS. POS5, which encodes a mitochondrial NAD kinase in Saccharomyces cerevisiae, is responsible for the de novo biosynthesis of NADPH in mitochondria. As a critical source of mitochondrial NADPH, Pos5p plays vital roles in various mitochondrial functions. However, some of the mitochondrial NADP+-dependent dehydrogenase, such as a mitochondrial NADP+-specific isocitrate dehydrogenase encoded by IDP1 gene, could also supply mitochondrial NADPH by the reducing of NADP+.In order to explore the predominant supplying pathway of mitochondrial NADPH, pos5idp1Δand pos5zwf1Δdouble mutant were constructed. Through the phenotypic study of pos5Δ, idp1Δ, pos5idp1Δand other double mutants, including their growth ability, temperature sensitivity, non-fermentable carbon sources usage ability, arginine and methionine biosynthetic ability and anti-oxidation, supplying mode of mitochondrial NADPH and function of NAD kinase in mitochondria were indicated. It is found that, Pos5p is the main supplier of mitochondrial NADPH and deletion of POS5 will result in various growth defects of strains, whereas Idp1p is not so important as Pos5p in the contribution of NADPH.Since the deletion of POS5 shows the growth defects on a non-fermentable carbon source, indicating that the deletion of POS5 may result in the defects in respiration, thus the activities of respiratory chain complex of BY4742, pos5Δ, idp1Δand pos5idp1Δgrowing at middle log phase and stationary phase in SD and YPD medium were measured, including that of Complex II (succinate dehydrogenase; EC 1.3.5.1), Complex III (cytochrome bc1 complex; EC 1.10.2.2), Complex IV (cytochrome c oxidase; EC 1.9.3.1) and Complex II+ III. Results showed that the respiratory chain activities of mutants are lower than that of BY4742, with the activity of pos5idp1Δthe lowest, which confirmed that the respiration defect of pos5Δactually has relation with the decrease of respiration chain activity.Pos5p is the main supplyer of NADPH in mitochondrial, which converts NAD(H) to NADP(H), whereas Zwf1p is the main supplyer of NADPH in cytoplasm, which converts NADP+ to NADPH. Conversion of the different forms of these coenzymes is the main reason for these two enzymes performing the particular function and giving the particular phenotype. In order to understand whether and how Pos5p and Zwf1p affect the form and content of coenzymes, intracellular content of NAD+, NADH, NADP+ and NADPH in different stains were analysized by HPLC. The result showed that the contents of NADP+ and NADPH in idp1Δ, pos5Δand pos5idp1Δare lower than that of BY4742 and pos5Δ/POS5-YEp, and the contents of NADP+ and NADPH in zwf1Δand pos5zwf1Δare the lowest.