Two Sources of Mitochondrial NADPH in the Yeast Saccharomyces cerevisiae

Hikaru Miyagi,Shigeyuki Kawai,Kousaku Murata

doi:10.1074/jbc.m804100200

Abstract

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.

Highlights

Exchange between NADH and NADPϩ or NADϩ and NADPH, resulting in the formation of NADPH from NADPϩ (1)
Lethality of Utr1yef1pos5—We previously reported that the S. cerevisiae NAD kinase triple mutant utr1yef1pos5 (MK1219: BY4742 utr1⌬::kanMX4 yef1⌬::HIS3 pos5⌬::CgLEU2) was viable (5)
This study has demonstrated that the mitochondrial NAD kinase reaction followed by the NADPϩ-dependent acetaldehyde dehydrogenase reaction catalyzed by Ald4/Ald5p, and in particular Ald4p, serves as a source of mitochondrial NADPH in S. cerevisiae

Summary

Mitochondrial NADPH in Yeast

The higher NADH kinase activity of Pos5p compared with its NAD kinase activity led Otten and Culotta (6, 13) to speculate that the NADH kinase reaction catalyzed by Pos5p, rather than the NAD kinase reaction, is critical to the supply of mitochondrial NADPH. These authors presented no evidence to corroborate this speculation, and neither were the kinetic values of Pos5p determined. The possibility remains that, in addition to the NAD kinase reaction catalyzed by Pos5p, any one of these other dehydrogenases may contribute physiologically to the supply of mitochondrial NADPH. The physiological significance of the NADH kinase reaction in the absence of Ald4p is demonstrated

EXPERIMENTAL PROCEDURES

Plasmids used in this study

RESULTS

DISCUSSION