J. Biol. Chem., Vol. 265, Issue 13, 7273-7277, May, 1990
Mitochondrial import of cytochrome c oxidase subunit VIIa in Saccharomyces cerevisiae. Identification of sequences required for mitochondrial localization in vivo
DM Duhl, T Powell and RO Poyton
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309-0347.
Subunit VIIa of yeast cytochrome c oxidase is a small (59 amino acids)
protein of the inner mitochondrial membrane that lacks a cleavable
amino-terminal presequence. To identify regions within this polypeptide
that are essential for its import, gene fusions were constructed using a
leader peptide substitution vector (pLPS) developed in this laboratory
(Glaser, S. M., Trueblood, C. E., Dircks, L. K., Poyton, R. O., and Cumsky,
M. G. (1988) J. Cell. Biochem. 36, 275-287). In this vector,
oligonucleotide sequences encoding all or part of subunit VIIa were fused
in-frame with the coding region of mature cytochrome c oxidase subunit Va.
The plasmid pLPS is ideal for assaying protein sequences for their ability
to direct mitochondrial import in vivo since subunit Va's leader peptide is
essential for import and because subunit V is required for cytochrome c
oxidase activity and respiration. Strains containing these fusions but
lacking both subunit V genes (COX5a and COX5b) were analyzed to determine
whether the chimeric protein is directed to mitochondria. Our findings
indicate that the amino-terminal 17 amino acids of subunit VIIa are
sufficient to localize subunit Va to the mitochondrion and that a 6-amino
acid- long region within the amino terminus (Gly8-Arg13) is essential. In
addition, some import (approximately 10% of wild type) is observed with the
highly charged carboxyl terminus of subunit VIIa, suggesting that the
subunit may contain redundancy in its import information.
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