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(Received for publication, May 14, 1996, and in revised form, May 24, 1996)
From the Biochemistry Department, School of Biological Sciences,
University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom
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.
Volume 271, Number 29,
Issue of July 19, 1996
pp. 17062-17066
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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