The essential function of the small Tim proteins in the TIM22 import pathway does not depend on formation of the soluble 70-kilodalton complex

The TIM22 protein import pathway of the yeast mitochondrion contains severa l 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 guid e hydrophobic precursors across the intermembrane space. Like membrane-asso ciated Tim12p, they are members of the Tim18p-Tim22p-Tim54p membrane comple x that mediates precursor insertion into the membrane. To understand the ro le of this family in protein import, we have used a genetic approach to man ipulate the complement of the small Tim proteins. A strain has been constru cted that lacks the 70-kDa soluble Tim8p-Tim13p and Tim9p-Tim10p complexes in the intermembrane space. Instead, a functional version of Tim9p (Tim9(S6 7C)p), identified as a second-site suppressor of a conditional tim10 mutant , maintains viability. Characterization of this strain revealed that Tim9(S 67C)p and Tim10p were tightly associated with the inner membrane, the solub le 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 Tim9(S67C)p was loca ted in the intermembrane space, associated with the inner membrane. We sugg est that the 70-kDa complexes facilitate import, similar to the outer membr ane receptors of the TOM (hetero-oligomeric translocase of the outer membra ne) complex, and the essential role of Tim9p and Tim10p may be to mediate p rotein insertion in the inner membrane with the TIM22 complex.