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 envelope1-5. 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 channel6-11. 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.

Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors.

Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive. Here we determined the structure of TOM core complex at 2.53 Å and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 Å. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain. Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.

Architecture and assembly dynamics of the essential mitochondrial chaperone complex TIM9·10·12.

Tim chaperones transport membrane proteins to the two mitochondrial membranes. TIM9·10, a 70 kDa protein complex formed by 3 copies of Tim9 and Tim10, guides its clients across the aqueous compartment. The TIM9·10·12 complex is the anchor point at the inner-membrane insertase TIM22. The subunit composition of TIM9·10·12 remains debated. Joint NMR, small-angle X-ray scattering, and MD simulation data allow us to derive a structural model of the TIM9·10·12 assembly, with a 2:3:1 stoichiometry (Tim9:Tim10:Tim12). Both TIM9·10 and TIM9·10·12 hexamers are in a dynamic equilibrium with their constituent subunits, exchanging on a minutes timescale. NMR data establish that the subunits exhibit large conformational dynamics: when the conserved cysteines of the CX3C-Xn-CX3C motifs are formed, short α helices are formed, and these are fully stabilized only upon formation of the mature hexameric chaperone. We propose that the continuous subunit exchange allows mitochondria to control their level of inter-membrane space chaperones.