MULTIPLE INTERACTIONS OF COMPONENTS MEDIATING PREPROTEIN TRANSLOCATION ACROSS THE INNER MITOCHONDRIAL-MEMBRANE

The protein transport machinery of the inner mitochondrial membrane co ntains three essential Tim proteins, Tim17 and Tim23 are thought to bu ild a preprotein translocation channel, while Tim44 transiently intera cts with the matrix heat shock protein Hsp70 to form an ATP-driven imp ort motor, For this report we characterized the biogenesis and interac tions of Tim proteins, (i) Import ol the precursor of Tim44 into the i nner membrane requires mtHsp70, whereas import and inner membrane inte gration of the precursors of Tim17 and Tim23 are independent of functi onal mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70 , but only weakly with Tim44, (iii) Depletion of Tim44 does not affect the co-precipitation of Tim17 with antibodies directed against mtHsp7 0, (iv) Tim23 associates with both Tim44 and Tim17, suggesting the pre sence of two Tim23 pools in the inner membrane, a Tim44-Tim23-containi ng subcomplex and a Tim23-Tim17-containing sub-complex. (v) The associ ation of mtHsp70 with the Tim23-Tim17 sub-complex is ATP sensitive and can be distinguished from the mtHsp70-Tim44 interaction by the differ ential influence of an amino acid substitution in mtHsp70. (vi) Geneti c evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditi onal mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transp ort machinery of the mitochondrial inner membrane consists of dynamica lly interacting sub-complexes, each of which transiently binds mtHsp70 .