contributed equally to this work Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.

Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.Most mitochondrial proteins are synthesized in the cytosol with a cleavable N-terminal presequence that specifies import into mitochondria via the general protein import pathway (25,27,32,36). This pathway is mediated by cytosolic chaperones, a hetero-oligomeric TOM complex in the mitochondrial outer membrane, a Tim17p-Tim23p complex (referred to as the TIM23 complex) in the inner membrane, an ATP-dependent import motor associated with the matrix face of the TIM23 complex, and soluble proteins in the matrix involved in the proteolytic maturation and folding of the imported proteins (9,11,(27)(28)(29).Over the past few years it has become clear that mitochondria possess an additional pathway which affects import of hydrophobic inner membrane proteins (17,22,27). This pathway diverges from the general import pathway after the TOM channel (23). As the hydrophobic precursor exits that channel, it is met by one of the two soluble 70-kDa protein complexes that transfer it across the intermembrane space (1,19,21,39). One of these 70-kDa complexes contains Tim9p and Tim10p, while the other contains Tim8p and Tim13p (20). Both complexes generally deliver hydrophobic proteins to an inner membrane complex specialized for the insertion of membrane proteins. This insertion complex, referred to as the TIM22 complex, has an apparent mass of 300 kDa and contains the membrane proteins Tim22p and Tim54p, the peripheral membrane protein Tim12p, and a small proportion of Tim9p and Tim10p (1,18,19,21,39). The 70-kDa Tim9p-Tim10p complex can also deliver some membrane proteins to the TIM23 complex and, possibly, to additional, as-yet-unknown insertion sites (24).A typical protein imported by ...