THE TIM CORE COMPLEX DEFINES THE NUMBER OF MITOCHONDRIAL TRANSLOCATION CONTACT SITES AND CAN HOLD ARRESTED PREPROTEINS IN THE ABSENCE OF MATRIX HSP70-TIM44

Preprotein import into mitochondria is mediated by translocases locate d in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Ti m44 driving system. By blue native electrophoresis, we identify an sim ilar to 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Pre proteins spanning the two membranes link virtually all Tim core comple xes with one in four Tom complexes in a stable 600K supercomplex. Neit her mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600 K complex. Preproteins in transit stabilize the Tim core complex, prev enting an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not p assive diffusion channels, but can stably interact with preproteins an d determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effec t on the Tim channel.