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Title: Structural basis of mitochondrial protein import by the TIM23 complex
Mitochondria import nearly all of their approximately 1,000–2,000 constituent proteins from the cytosol across their double-membrane envelope1,2,3,4,5. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel6,7,8,9,10,11. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy 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 the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has 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.  more » « less
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National Science Foundation
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  1. Background

    The translocase of the mitochondrial inner membrane (TIM) imports most of the nucleus‐encoded proteins that are destined for the matrix, inner membrane (IM) and the intermembrane space (IMS).Trypanosoma brucei, the infectious agent for African trypanosomiasis, possesses a unique TIM complex consisting of several novel proteins in association with a relatively conserved protein TbTim17. Tandem affinity purification of the TbTim17 protein complex revealed TbTim54 as a potential component of this complex.


    TbTim54, a trypanosome‐specific IMS protein, is peripherally associated with the IM and is present in a protein complex slightly larger than the TbTim17 complex. TbTim54 knockdown (KD) reduced the import of TbTim17 and compromised the integrity of the TbTim17 complex. TbTim54 KD inhibited thein vitromitochondrial import and assembly of the internal signal‐containing mitochondrial carrier proteins MCP3, MCP5 and MCP11 to a greater extent than TbTim17 KD. Furthermore, TbTim54 KD, but not TbTim17 KD, significantly hampered the mitochondrial targeting of ectopically expressed MCP3 and MCP11. These observations along with our previous finding that the mitochondrial import of N‐terminal signal‐containing proteins like cytochrome oxidase subunit 4 and MRP2 was affected to a greater extent by TbTim17 KD than TbTim54 KD indicating a substrate‐specificity of TbTim54 for internal‐signal containing mitochondrial proteins. In other organisms, small Tim chaperones in the IMS are known to participate in the translocation of MCPs. We found that TbTim54 can directly interact with at least two of the six known small TbTim proteins, TbTim11 and TbTim13, as well as with the N‐terminal domain of TbTim17.


    TbTim54 interacts with TbTim17. It also plays a crucial role in the mitochondrial import and complex assembly of internal signal‐containing IM proteins inT. brucei.


    We are the first to characterise TbTim54, a novel TbTim that is involved primarily in the mitochondrial import of MCPs and TbTim17 inT. brucei.

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