The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.

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Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. This complex consists of receptor components for the initial contact with preproteins at the mitochondrial surface and membrane-embedded proteins which promote transport and form the translocation pore. In order to understand the interplay between the translocating preprotein and the constituents of the TOM complex, we analyzed the dynamics of the TOM complex of Neurospora crassa and Saccharomyces cerevisiae mitochondria by following the structural alterations of the essential pore component Tom40 during the translocation of preproteins. Tom40 exists in a homo-oligomeric assembly and dynamically interacts with Tom6. The Tom40 assembly is influenced by a block of negatively charged amino acid residues in the cytosolic domain of Tom22, indicating a cross-talk between preprotein receptors and the translocation pore. Preprotein binding to specific sites on either side of the outer membrane (cis and trans sites) induces distinct structural alterations of Tom40. To a large extent, these changes are mediated by interaction with the mitochondrial targeting sequence. We propose that such targeting sequence-induced adaptations are a critical feature of translocases in order to facilitate the movement of preproteins across cellular membranes.The import of proteins into mitochondria is mediated by multisubunit translocases in the outer (TOM complex) and inner (TIM complex) membranes of the organelles (23,28,33). The TOM complex contains components which expose domains to the cytosol and act as preprotein receptors. The major import receptors are Tom20 and Tom22, which are essential for the specific recognition, unfolding, and translocation of the majority of preproteins (22). Both components interact with preproteins and cooperate in the formation of a presequence binding site termed the cis site (3,20,25,26,34). Another binding site for a more restricted set of preproteins, especially for members of the mitochondrial carrier family, is Tom70 (13,35,36), which acts in conjunction with Tom37 (12). From this binding site, preproteins are transferred to Tom20-Tom22 before entering the translocation pore (19).Other components of the TOM complex (Tom40, Tom5, Tom6, and Tom7) are deeply embedded in the outer membrane and are believed to form the translocation pore. Tom40 is an essential protein and was found in the vicinity of polypeptide chains in transit (31, 37, 39). The protein was suggested to be a central element of the preprotein-conducting pore of the mitochondrial outer membrane. The small members of the TOM complex are not essential by themselves, but combined deletion of their genes and those of other components of the translocase is lethal (1, 6, 15). Studies on the function of the small TOM complex proteins suggest that they play distinct roles. Tom6 and Tom7 were found to influence the stability of the TOM complex (1, 15). For Tom5 a function in facilitating preprotein transfer from the receptors into the translocati...

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound ef®ciently in a speci®c manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using¯uorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The af®nity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the translocase of the outer mitochondrial membrane (TOM) complex. We report the molecular identification of Tom6 and Tom7, two small subunits of the TOM core complex in the fungus Neurospora crassa. Cross-linking experiments showed that both proteins were found to be in direct contact with the major component of the pore, Tom40. In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit. Precursors of both proteins are able to insert into the outer membrane in vitro and are assembled into authentic TOM complexes. The insertion pathway of these proteins shares a common binding site with the general import pathway as the assembly of both Tom6 and Tom7 was competed by a matrix-destined precursor protein. This assembly was dependent on the integrity of receptor components of the TOM machinery and is highly specific as in vitro-synthesized yeast Tom6 was not assembled into N. crassa TOM complex. The targeting and assembly information within the Tom6 sequence was found to be located in the transmembrane segment and a flanking segment toward the N-terminal, cytosolic side. A hybrid protein composed of the C-terminal domain of yeast Tom6 and the cytosolic domain of N. crassa Tom6 was targeted to the mitochondria but was not taken up into TOM complexes. Thus, both segments are required for assembly into the TOM complex. A model for the topogenesis of the small Tom subunits is discussed.