Dynamics of the TOM Complex of Mitochondria during Binding and Translocation of Preproteins

Abstract

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...

Assembly of Tom6 and Tom7 into the TOM Core Complex ofNeurospora crassa

“…Tom7 is in direct contact with Tom40, whereas the contact of Tom6 with Tom22 and Tom40 is dynamically modulated by preproteins in transit. Because chemical cross-linking did not reveal a direct contact between Tom40 and Tom22 (not shown), these results provide experimental support to the previously suggested role of Tom6 as a linking component between Tom40 and Tom22 (10,25).…”

“…The information for assembly of Tom6 into the complex is most likely located at the N-terminal flanking region of the putative transmembrane segment. A proteolytic fragment of Tom6 that lacked few N-terminal residues of its cytosolic domain maintained the ability to interact with Tom40 (25). Furthermore, deletion of the N-terminal 12 amino acid residues did not affect assembly of the resulting construct, whereas deletion of 36 of 38 residues of the cytosolic domain resulted in an assembly-incompetent precursor.…”

“…Tom6 and Tom7 Are in Direct Contact with Other Members of the TOM Core Complex-Tom6 was previously found to be in the vicinity of Tom40, and their interaction was modified by the formation of specific translocation intermediates of a precursor protein (25). To determine whether both Tom6 and Tom7 are only in the vicinity of Tom40 or actually in direct contact with the protein, the cross-linking reagent EDC was added to outer membrane vesicles, and cross-linking products were analyzed by immunodecoration.…”

Protein Import Into Mitochondria

“…The intermembrane space domain of Tom22 may also contribute to trans-site binding of presequences (63,64). Trans-site binding occurs with much higher af nity then cis-site binding: the measured K d 's were in the low ¹molar range (50).…”

“…These pores presumably represent the protein-conducting channels. Given that the TOM complex contains approximately eight Tom40 molecules, one pore might be formed by two Tom40 dimers (50). However, electron microscopy of puri ed Tom40 revealed particles mainly with one pore con rming that Tom40 forms the protein-conducting channel in an oligomeric assembly (45).…”