Two Sources of Mitochondrial NADPH in the Yeast Saccharomyces cerevisiae*

Cells of the yeast Saccharomyces cerevisiae contain three NAD kinases; namely, cytosolic Utr1p, cytosolic Yef1p, and mitochondrial Pos5p. Previously, the NADH kinase reaction catalyzed by Pos5p, rather than the NAD kinase reaction followed by the NADP+-dependent dehydrogenase reaction, had been regarded as a critical source of mitochondrial NADPH, which plays vital roles in various mitochondrial functions. This study demonstrates that the mitochondrial NADH kinase reaction is dispensable as a source of mitochondrial NADPH and emphasizes the importance of the NAD kinase reaction, followed by the mitochondrial NADP+-dependent dehydrogenase reaction. Of the potential dehydrogenases (malic enzyme, Mae1p; isocitrate dehydrogenase, Idp1p; and acetaldehyde dehydrogenases, Ald4/5p), evidence is presented that acetaldehyde dehydrogenases, and in particular Ald4p, play a prominent role in generating mitochondrial NADPH in the absence of the NADH kinase reaction. The physiological significance of the mitochondrial NADH kinase reaction in the absence of Ald4p is also demonstrated. In addition, Pos5p is confirmed to have a considerably higher NADH kinase activity than NAD kinase activity. Taking these results together, it is proposed that there are two sources of mitochondrial NADPH in yeast: one is the mitochondrial Pos5p-NADH kinase reaction and the other is the mitochondrial Pos5p-NAD kinase reaction followed by the mitochondrial NADP+-dependent acetaldehyde dehydrogenase reaction.

[1]  B. Mikami,et al.  Molecular Conversion of NAD Kinase to NADH Kinase through Single Amino Acid Residue Substitution* , 2005, Journal of Biological Chemistry.

[2]  S. Brunak,et al.  Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. , 2000, Journal of molecular biology.

[3]  G. Natsoulis,et al.  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. , 1987, Methods in enzymology.

[4]  S. Kawai,et al.  Identification of ATP‐NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae , 2005, The FEBS journal.

[5]  L. McAlister-Henn,et al.  Sources of NADPH in Yeast Vary with Carbon Source* , 2005, Journal of Biological Chemistry.

[6]  S. Kawai,et al.  Molecular characterization of Escherichia coli NAD kinase. , 2001, European journal of biochemistry.

[7]  E. Noltmann,et al.  Glucose 6-phosphate dehydrogenase (Zwischenferment). I. Isolation of the crystalline enzyme from yeast. , 1961, The Journal of biological chemistry.

[8]  G. R. Stuart,et al.  POS5 Gene of Saccharomyces cerevisiae Encodes a Mitochondrial NADH Kinase Required for Stability of Mitochondrial DNA , 2003, Eukaryotic Cell.

[9]  Charles Brenner,et al.  Synthetic Lethal and Biochemical Analyses of NAD and NADH Kinases in Saccharomyces cerevisiae Establish Separation of Cellular Functions* , 2006, Journal of Biological Chemistry.

[10]  K. Pawan,et al.  Defect in oxidative phosphorylation in LV papillary muscle mitochondria of patients undergoing mitral valve replacement. , 2006, Mitochondrion.

[11]  Y. Surdin-Kerjan,et al.  Identification of the structural gene for glucose‐6‐phosphate dehydrogenase in yeast. Inactivation leads to a nutritional requirement for organic sulfur. , 1991, The EMBO journal.

[12]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[13]  R. Haselbeck,et al.  Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. , 1991, The Journal of biological chemistry.

[14]  M. Ziegler,et al.  The power to reduce: pyridine nucleotides--small molecules with a multitude of functions. , 2007, The Biochemical journal.

[15]  J. Turrens.  Superoxide Production by the Mitochondrial Respiratory Chain , 1997, Bioscience reports.

[16]  B. Mackler,et al.  Pyridine nucleotide transhydrogenations in yeast. , 1985, Archives of biochemistry and biophysics.

[17]  G. Daum,et al.  Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. , 1982, The Journal of biological chemistry.

[18]  G. Jennings,et al.  Sources of NADPH and Expression of Mammalian NADP+-specific Isocitrate Dehydrogenases inSaccharomyces cerevisiae * , 1998, The Journal of Biological Chemistry.

[19]  S. Dequin,et al.  Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. , 2004, Microbiology.

[20]  W. Snedden,et al.  Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress). , 2005, The Biochemical journal.

[21]  Su-Min Lee,et al.  Control of Mitochondrial Redox Balance and Cellular Defense against Oxidative Damage by Mitochondrial NADP+-dependent Isocitrate Dehydrogenase* , 2001, The Journal of Biological Chemistry.

[22]  P. Philippsen,et al.  Heterologous HIS3 Marker and GFP Reporter Modules for PCR‐Targeting in Saccharomyces cerevisiae , 1997, Yeast.

[23]  V. Culotta,et al.  Cellular factors required for protection from hyperoxia toxicity in Saccharomyces cerevisiae. , 2005, The Biochemical journal.

[24]  E. O’Shea,et al.  Global analysis of protein expression in yeast , 2003, Nature.

[25]  D. Kosman,et al.  The Yeast Copper/Zinc Superoxide Dismutase and the Pentose Phosphate Pathway Play Overlapping Roles in Oxidative Stress Protection* , 1996, The Journal of Biological Chemistry.

[26]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[27]  S. Kawai,et al.  Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp. A1. , 2004, Protein expression and purification.

[28]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[29]  J. Pronk,et al.  Identification and Characterization ofMAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme , 1998, Journal of bacteriology.

[30]  M. Ziegler,et al.  NAD Kinase Levels Control the NADPH Concentration in Human Cells* , 2007, Journal of Biological Chemistry.

[31]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[32]  S. Kawai,et al.  Molecular cloning and identification of UTR1 of a yeast Saccharomyces cerevisiae as a gene encoding an NAD kinase. , 2001, FEMS microbiology letters.

[33]  Dorota Grabowska,et al.  The ALD6 Gene Product Is Indispensable for Providing NADPH in Yeast Cells Lacking Glucose-6-phosphate Dehydrogenase Activity* , 2003, The Journal of Biological Chemistry.

[34]  V. Culotta,et al.  A novel NADH kinase is the mitochondrial source of NADPH in Saccharomyces cerevisiae , 2003, The EMBO journal.

[35]  J. Mccusker,et al.  Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae , 1999, Yeast.