Modular structure of the TIM23 preprotein translocase of mitochondria

The TIM23 complex mediates import into mitochondria of nuclear encoded prep roteins with a matrix-targeting signal. It is composed of the integral memb rane proteins Tim17 and Tim23 and the peripheral membrane protein Tim44, wh ich recruits mitochondrial Hsp70 to the sites of protein import. We have an alyzed the functions of these constituents using a combined genetic and bio chemical approach. Depletion of either Tim17 or Tim23 led to loss of import competence of mitochondria and to a reduction in the number of preprotein- conducting channels. Upon depletion of Tim44, mitochondria also lost their ability to import proteins but maintained normal numbers of import channels . In the absence of Tim44 precursor protein was specifically recognized. Th e presequence was translocated in a Delta psi -dependent manner across the inner membrane and cleaved by matrix-processing peptidase, However, the pre protein did not move further into the matrix but rather underwent retrograd e sliding out of the TIM23 complex. Thus, the TIM23 complex is composed of functionally independent modules. Tim17 and Tim23 are necessary for initiat ing translocation, whereas Tim44 and mitochondrial Hsp70 are indispensable for complete transport of preproteins and for unfolding of folded domains o f preproteins.