Abstract #97A

Role of the cytochrome c oxidase subunit II C-terminal domain in membrane insertion and translocation to the mitochondrial intermembrane space. Heather L. Fiumera, Sarah A. Broadley, Thomas D. Fox. Dept Molecular Biol & Genetics, Cornell Univ, Ithaca, NY 14853.
   Assembly of cytochrome c oxidase requires both the N- and C-terminal hydrophilic domains of mitochondrially synthesized Cox2p to be translocated through the inner membrane to the inter membrane space (IMS), while two transmembrane helices are inserted into the membrane. In yeast, the activities of nuclearly encoded inner membrane proteins are required for this translocation, including Oxa1p for N-tail and C-tail export, and Mss2p and Cox18p for C-tail export. Oxa1p and Cox18p are functionally conserved in humans. We find that Mss2p, a peripheral protein on the inner surface of the inner membrane, interacts with newly synthesized Cox2p-HA3 by co-immunoprecipitation, but not with assembled Cox2p-HA3. Thus, Mss2p likely has a direct role in translocation. A truncated protein lacking the C-tail domain, Cox2(1-109)-HA3, does not interact with Mss2p, suggesting that the 144 residue acidic C-tail may contact Mss2p. Consistent with the possibility that recognition of the Cox2p C-tail by Mss2p (and/or other factors) is required for translocation, removal of the last 40 amino acids of the C-tail domain prevents export of an epitope at the end of the truncated protein (Cox2p(1-211)-HA3). The copper binding domain present in these last 40 residues is not required for translocation, since mutations destroying this site do not prevent export of the C-terminus. The truncated Cox2p(1-109)-HA3, lacking the C-tail domain, is unstable in vivo, but the protein that does accumulate has both N- and C- termini exported to the IMS. Export of both termini of the truncated protein is blocked in the absence of Oxa1p. However, in the absence of either Cox18p or Mss2p, most of the truncated protein accumulates with its N- and C-termini in the IMS. Thus, removal of the acidic C-tail domain allows the second Cox2p transmembrane domain to insert across the inner membrane, carrying HA epitopes with it, independently of Cox18p and Mss2p.

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