The Essential Function of the Small Tim Proteins in the TIM22 Import Pathway Does Not Depend on Formation of the Soluble 70-Kilodalton Complex

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Abstract
The TIM22 protein import pathway of the yeast mitochondrion contains several components, including a family of five proteins (Tim8p, -9p, -10p, -12p, and -13p [Tim, for translocase of inner membrane]) that are located in the intermembrane space and are 25% identical. Tim9p and Tim10p have dual roles in mediating the import of inner membrane proteins. Like the Tim8p-Tim13p complex, the Tim9p-Tim10p complex functions as a putative chaperone to guide hydrophobic precursors across the intermembrane space. Like membrane-associated Tim12p, they are members of the Tim18p-Tim22p-Tim54p membrane complex that mediates precursor insertion into the membrane. To understand the role of this family in protein import, we have used a genetic approach to manipulate the complement of the small Tim proteins. A strain has been constructed that lacks the 70-kDa soluble Tim8p-Tim13p and Tim9p-Tim10p complexes in the intermembrane space. Instead, a functional version of Tim9p (Tim9S67Cp), identified as a second-site suppressor of a conditional tim10 mutant, maintains viability. Characterization of this strain revealed that Tim9S67Cp and Tim10p were tightly associated with the inner membrane, the soluble 70-kDa Tim8p-Tim13p and Tim9p-Tim10p complexes were not detectable, and the rate of protein import into isolated mitochondria proceeded at a slower rate. An arrested translocation intermediate bound to Tim9S67Cp was located in the intermembrane space, associated with the inner membrane. We suggest that the 70-kDa complexes facilitate import, similar to the outer membrane receptors of the TOM (hetero-oligomeric translocase of the outer membrane) complex, and the essential role of Tim9p and Tim10p may be to mediate protein insertion in the inner membrane with the TIM22 complex.