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      Two Intermembrane Space Tim Complexes Interact with Different Domains of Tim23p during Its Import into Mitochondria

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          Abstract

          Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p–Tim13p and Tim9p–Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p–Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH 2-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p–Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p–Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.

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          A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels.

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            The preprotein translocation channel of the outer membrane of mitochondria.

            The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.
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              Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22.

              Translocation of mitochondrial preproteins across the inner membrane is facilitated by the TIM machinery. Tim23 binds to matrix targeting signals and initiates membrane potential-dependent import. Tim23 and Tim17 are constituents of a translocation channel across the inner membrane. Tim44 is associated with this channel at the matrix side, and Tim44 recruits mitochondrial Hsp70 and its co-chaperone Mgel, which drive protein translocation into the matrix using ATP as an energy source. Tim22 is a new component of the import machinery of mitochondria, which shares sequence similarity with both Tim23 and Tim17. Here we report that Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Members of the yeast AAC family are synthesized without matrix targeting signals. Tim22 is in an assembly of high relative molecular mass that is distinct from the Tim23-Tim17 complex. Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent of Tim22.
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                Author and article information

                Contributors
                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                18 September 2000
                : 150
                : 6
                : 1271-1282
                Affiliations
                [a ]Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
                [b ]Department of Medical Biochemistry and Genetics, Texas A&M University Health Science Center, College Station, Texas 77843
                [c ]Department of Chemistry, Texas A&M University Health Science Center, College Station, Texas 77843
                [d ]Department of Biochemistry and Biophysics, Texas A&M University Health Science Center, College Station, Texas 77843
                Article
                0004057
                2150706
                10995434
                7c01d91d-eddf-4c09-aed3-a586eb9755d5
                © 2000 The Rockefeller University Press
                History
                : 13 April 2000
                : 2 August 2000
                : 4 August 2000
                Categories
                Original Article

                Cell biology
                cross-linking,protein translocation
                Cell biology
                cross-linking, protein translocation

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