We have identified a new protein, Tim54p, located in the yeast mitochondrial inner membrane. Tim54p is an essential import component, required for the insertion of at least two polytopic proteins into the inner membrane, but not for the translocation of precursors into the matrix. Several observations suggest that Tim54p and Tim22p are part of a protein complex in the inner membrane distinct from the previously characterized Tim23p-Tim17p complex. First, multiple copies of the TIM22 gene, but not TIM23 or TIM17, suppress the growth defect of a tim54-1 temperature-sensitive mutant. Second, Tim22p can be coprecipitated with Tim54p from detergent-solubilized mitochondria, but Tim54p and Tim22p do not interact with either Tim23p or Tim17p. Finally, the tim54-1 mutation destabilizes the Tim22 protein, but not Tim23p or Tim17p. Our results support the idea that the mitochondrial inner membrane carries two independent import complexes: one required for the translocation of proteins across the inner membrane (Tim23p–Tim17p), and the other required for the insertion of proteins into the inner membrane (Tim54p–Tim22p).

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Tim23p is a mitochondrial inner membrane protein essential for the import of proteins from the cytosol. Tim23p contains an amino-terminal hydrophilic segment and a carboxyl-terminal hydrophobic domain (Tim23Cp). To study the functions and interactions of the two parts of Tim23p separately, we constructed tim23N, encoding only the hydrophilic region of Tim23p, and tim23C, encoding only the hydrophobic domain of Tim23p. Only the Tim23C protein is imported into mitochondria, indicating that the mitochondrial targeting information in Tim23p resides in its membrane spans or intervening loops. Tim23Cp, however, cannot substitute for full-length Tim23p, suggesting that the hydrophilic portion of Tim23p also performs an essential function in mitochondrial protein import. We found that overexpression of Tim23Cp is toxic to yeast cells that carry the tim23-1 mutation. Excess Tim23Cp causes Tim23-1p to disappear, leaving tim23-1 cells without a full-length version of the Tim23 protein. If Tim17p, another inner membrane import component, is overexpressed along with Tim23Cp, the toxicity of Tim23Cp is largely reversed and the Tim23-1 protein no longer disappears. In coimmunoprecipitations from solubilized mitochondria, Tim17p associates with the Tim23C protein. In addition, we show that Tim23p and Tim17p can be chemically cross-linked to each other in intact mitochondria. We conclude that the hydrophobic domain encoded by tim23C targets Tim23p to the mitochondria and mediates the direct interaction between Tim23p and Tim17p. In contrast, Tim23Cp cannot be coimmunoprecipitated with Tim23p, raising the possibility that the hydrophobic domain of Tim23p does not interact with other Tim23 molecules.Most mitochondrial proteins are encoded in the nucleus, translated in the cytosol, targeted to the mitochondria, and then translocated across one or both mitochondrial membranes to their final destinations (reviewed in references 50, 56, 59, and 60). Mitochondrial proteins are usually synthesized as precursors, with positively charged amino-terminal presequences that contain targeting information (23)(24)(25)74). On the mitochondrial surface, precursors encounter several proteins proposed to act as receptors, including Tom70p, Tom37p, Tom22p, and Tom20p (16,21,31,44,62,68,69). Cytosolic chaperones bind precursors to prevent premature folding or aggregation, and one chaperone also plays a role in targeting the precursor to the mitochondrial surface (17,18,34). The outer membrane receptors, along with several other proteins, including Tom40p, Tom6p, Tom7p, and Tom8p, make up the TOM complex, which translocates precursors across the mitochondrial outer membrane (32,33,46,70). Translocation of the precursor across the outer membrane is proposed to occur via a transfer of the presequence from a binding site on the outside of the mitochondria to a site located on the inside of the membrane (45). The interaction between the positively charged presequence and the TOM components appears to be at least partly electrostatic (6, 45).At least thre...