Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe.

We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1. 14 x 10(8) to 9.12 x 10(7) cells ml-1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth.

A highly conserved glutamate residue (Glu-270) is essential for plant alternative oxidase activity.

We have previously demonstrated that expression of a Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe confers cyanide-resistant respiratory activity on these cells (Albury, M. S., Dudley, P., Watts, F. Z., and Moore, A. L. (1996) J. Biol. Chem. 271, 17062-17066). Using this functional expression system we have investigated the active site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center. Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand within the active site, to asparagine abolishes catalytic activity because mitochondria containing the E270N mutant protein do not exhibit antimycin A-resistant respiration. Western blot analysis, using antibodies specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed by S. pombe mitochondria in a manner similar to that of the wild-type protein. It is possible that lack of antimycin A-insensitive respiration observed in mitochondria containing the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane. However, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lack of insertion is unlikely. These results provide the first experimental evidence to support the structural model in which the active site of the alternative oxidase contains a coupled binuclear iron center.

Targeting the plant alternative oxidase protein to Schizosaccharomyces pombe mitochondria confers cyanide-insensitive respiration.

The Sauromatum guttatum alternative oxidase has been expressed in Schizosaccharomyces pombe under the control of the thiamine-repressible nmt1 promoter. Alternative oxidase protein and activity were detected both in spheroplasts and isolated mitochondria, indicating that the enzyme is expressed in a functional form and confers cyanide-resistant respiration to S. pombe, which is sensitive to inhibition by octyl-gallate. Protein import studies revealed that the precursor form of the alternative oxidase protein is efficiently imported into isolated mitochondria and processed to its mature form comparable to that observed with potato mitochondria. Western blot analysis and respiratory studies revealed that the alternative oxidase protein is expressed in the inner mitochondrial membrane in its reduced (active) form. Treatment of mitochondria with diamide and dithiothreitol resulted in interconversion of the reduced and oxidized species and modulation of respiratory activity. The addition of pyruvate did not effect either the respiratory rate or expression of the reduced species of the protein. To our knowledge this is the first time that the alternative oxidase has been effectively targeted to and integrated into the inner mitochondrial membrane of S. pombe, and we conclude that the expression of a single polypeptide is sufficient for alternative oxidase activity.