Translocation of Mitochondrially Synthesized Cox2 Domains from the Matrix to the Intermembrane Space

Abstract

The N-terminal and C-terminal domains of mitochondrially synthesized cytochrome c oxidase subunit II, Cox2, are translocated through the inner membrane to the intermembrane space (IMS). We investigated the distinct mechanisms of N-tail and C-tail export by analysis of epitope-tagged Cox2 variants encoded in Saccharomyces cerevisiae mitochondrial DNA. Both the N and C termini of a truncated protein lacking the Cox2 C-terminal domain were translocated to the IMS via a pathway dependent upon the conserved translocase Oxa1. The topology of this Cox2 variant, accumulated at steady state, was largely but not completely unaffected in mutants lacking proteins required for export of the C-tail domain, Cox18 and Mss2. C-tail export was blocked by truncation of the last 40 residues from the C-tail domain, indicating that sequence and/or structural features of this domain are required for its translocation. Mss2, a peripheral protein bound to the inner surface of the inner membrane, coimmunoprecipitated with full-length newly synthesized Cox2, whose leader peptide had already been cleaved in the IMS. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus after the N-tail has been translocated by Oxa1.

We thank L. Briggs for technical assistance, as well as S. Saracco, S. Shabalina, N. Bsat, C. Martin, T. L. Mason, and G. Schatz for gifts of biological materials and reagents.

This study was supported by a postdoctoral fellowship to H.L.F. (GM65664) and a research grant to T.D.F. (GM29362) from the U.S. National Institutes of Health.