Structural basis of mitochondrial protein import by the TIM complex

Abstract

AbstractMitochondria 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 TIM complex (also known as TIM23 complex), mediates import of presequence-containing proteins into the mitochondrial matrix and inner membrane. Among ∼10 different subunits of the complex, the essential multi-pass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel. However, the mechanism of TIM-mediated protein import remains uncertain due to a lack of structural information on the complex. Here, we have determined the cryo-EM structure of the core TIM complex (Tim17–Tim23–Tim44) from Saccharomyces cerevisiae. We show that, contrary to the prevailing model, Tim23 and Tim17 do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Remarkably, our data suggest that the cavity of Tim17 itself forms the protein translocation path whereas Tim23 plays a structural role. We also show how the Tim17–Tim23 heterodimer associates with the scaffold protein Tim44 and J-domain proteins to mediate Hsp70-driven polypeptide transport into the matrix. Our work provides the structural foundation to understand the mechanism of TIM-mediated protein import and sorting, a central pathway in mitochondrial biogenesis.