The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox shuttle in Saccharomyces cerevisiae

Abstract

NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly, NDI1 is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogenases. Deletion of ADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures. Also, an ndi1Delta mutant was capable of respiratory growth under these conditions. However, when both ADH3 and NDI1 were deleted, metabolism became respirofermentative, indicating that the ethanol-acetaldehyde shuttle is essential for respiratory growth of the ndi1 delta mutant. In anaerobic batch cultures, the maximum specific growth rate of the adh3 delta mutant (0.22 h(-1)) was substantially reduced compared to that of the wild-type strain (0.33 h(-1)). This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions. Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in the adh3 delta ndi1 delta mutant, contributing to residual redox-shuttle activity in this strain.

FIG. 1

A scheme of the respiratory chain of Saccharomyces cerevisiae. Adh, alcohol dehydrogenase; bc1, bc₁ complex; cox, cytochrome c oxidase; Gpd, soluble glycerol-3-phosphate dehydrogenase; Gut2, membrane-bound glycerol-3-phosphate dehydrogenase; Nde, external NADH dehydrogenase; Ndi1, internal NADH dehydrogenase; Q, ubiquinone; G3P, glycerol-3-phosphate; DHAP, dihydroxy acetone phosphate.

FIG. 2

Proposed physiological role of the ethanol-acetaldehyde redox shuttle under anaerobic conditions (23). In the biosynthesis of amino acids, mitochondrial NADH is generated. Under anaerobic conditions, this NADH may be reoxidized after being shuttled to the cytosol, via formation of glycerol. Pdh, pyruvate-dehydrogenase complex; CS, citrate synthase; Idh, isocitrate dehydrogenase; Gdh, glutamate dehydrogenase.

FIG. 3

Physiology of S. cerevisiae CEN.PK209-1B (ndi1Δ) and CEN.PK113-7D (wild type) in aerobic, glucose-limited chemostat cultures. The steady-state biomass yield on glucose (Y_XS) and the specific rates of ethanol (q_ethanol), glycerol (q_glycerol), CO₂ (q_CO2), and O₂ (q_O2) production were measured for the ndi1Δ mutant (solid lines, closed symbols) and for the wild-type CEN.PK113-7D (dashed lines, open symbols) at various dilution rates. At dilution rates of 0.35 h⁻¹ or higher, the ndi1Δ strain washed out of the cultures and no steady state was reached. DW, dry weight.