Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane 1996

is not a static protein complex. Several sub-complexes The protein transport machinery of the inner mitocan be distinguished that assemble and dissociate in a chondrial membrane contains three essential Tim dynamic fashion (Alconada et al., 1995b; Gratzer et al., proteins. Tim17 and Tim23 are thought to build a 1995; Mayer et al., 1995; Bömer et al., 1996a; Haucke preprotein translocation channel, while Tim44 et al., 1996; Hönlinger et al., 1996). transiently interacts with the matrix heat shock The preprotein translocase of the inner mitochondrial protein Hsp70 to form an ATP-driven import motor. membrane (Tim) can operate independently of the Tom For this report we characterized the biogenesis and machinery. This was shown by the direct import of interactions of Tim proteins. (i) Import of the precursor preproteins across the inner membrane of mitochondria of Tim44 into the inner membrane requires mtHsp70, with a ruptured outer membrane (Ohba and Schatz, 1987; whereas import and inner membrane integration of Hwang et al., 1989). The Tom and Tim machineries can the precursors of Tim17 and Tim23 are independent be transiently connected via a precursor polypeptide in of functional mtHsp70. (ii) Tim17 efficiently associates transit (Horst et al., 1995). Three essential proteins of with Tim23 and mtHsp70, but only weakly with Tim44. the Tim machinery have been identified in the yeast (iii) Depletion of Tim44 does not affect the co-precipitaSaccharomyces cerevisiae: Tim44 (Maarse et al., 1992; tion of Tim17 with antibodies directed against Scherer et al., 1992), Tim23 (Dekker et al., 1993; Emtage mtHsp70. (iv) Tim23 associates with both Tim44 and and Jensen, 1993) and Tim17 (Maarse et al., 1994; Ryan Tim17, suggesting the presence of two Tim23 pools in et al., 1994) (the new uniform nomenclature for the the inner membrane, a Tim44–Tim23-containing subproteins is described in Pfanner et al., 1996). Tim17 and complex and a Tim23–Tim17-containing sub-complex. Tim23 are integral membrane proteins, whereas Tim44 (v) The association of mtHsp70 with the Tim23–Tim17 behaves as a peripheral membrane protein that is mainly sub-complex is ATP sensitive and can be distinguished located on the matrix side of the inner membrane. All from the mtHsp70–Tim44 interaction by the differenthree Tim proteins have been shown to be in close contact tial influence of an amino acid substitution in mtHsp70. with a preprotein in transit by the use of cross-linking (vi) Genetic evidence, suppression of the protein import reagents (Scherer et al., 1992; Blom et al., 1993; Horst defect of a tim17 yeast mutant by overexpression of et al., 1993; Ryan and Jensen, 1993; Kübrich et al., 1994; mtHsp70 and synthetic lethality of conditional mutants Berthold et al., 1995). in the genes of Tim17 and mtHsp70, supports a funcThe initial stage of transport of a preprotein into or tional interaction of mtHsp70 with Tim17. We conclude across the inner membrane is driven by the membrane that the protein transport machinery of the mitopotential Δψ, possibly by exerting an electrophoretic effect chondrial inner membrane consists of dynamically on the positively charged presequence (Martin et al., interacting sub-complexes, each of which transiently 1991). The unfolded precursor polypeptide chain emerging binds mtHsp70. on the matrix side is then bound by mtHsp70, an abundant