Cryo-EM and photocrosslinking studies reveal the mechanism of mitochondrial protein import by the TIM complex.

Abstract

Mitochondria are essential organelles that provide cells with energy through oxidative phosphorylation, as well as with many important metabolites. Mitochondria import nearly all their ∼1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing precursor proteins (preproteins) into the mitochondrial matrix and inner membrane. However, the mechanism by TIM23 forms a translocation path in the membrane and enables the import process remained unclear. We have determined the cryo-EM structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that surprisingly, the cavity of Tim17, not Tim23, forms the protein translocation path whereas Tim23 is likely to play a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.