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Review
. 2022 May;298(5):101870.
doi: 10.1016/j.jbc.2022.101870. Epub 2022 Mar 26.

Mitochondrial protein translocation machinery: From TOM structural biogenesis to functional regulation

Ulfat Mohd Hanif Sayyed  1 , Radhakrishnan Mahalakshmi  2
Affiliations
Review

Mitochondrial protein translocation machinery: From TOM structural biogenesis to functional regulation

Ulfat Mohd Hanif Sayyed et al. J Biol Chem. 2022 May.

Abstract

The human mitochondrial outer membrane is biophysically unique as it is the only membrane possessing transmembrane β-barrel proteins (mitochondrial outer membrane proteins, mOMPs) in the cell. The most vital of the three mOMPs is the core protein of the translocase of the outer mitochondrial membrane (TOM) complex. Identified first as MOM38 in Neurospora in 1990, the structure of Tom40, the core 19-stranded β-barrel translocation channel, was solved in 2017, after nearly three decades. Remarkably, the past four years have witnessed an exponential increase in structural and functional studies of yeast and human TOM complexes. In addition to being conserved across all eukaryotes, the TOM complex is the sole ATP-independent import machinery for nearly all of the ∼1000 to 1500 known mitochondrial proteins. Recent cryo-EM structures have provided detailed insight into both possible assembly mechanisms of the TOM core complex and organizational dynamics of the import machinery and now reveal novel regulatory interplay with other mOMPs. Functional characterization of the TOM complex using biochemical and structural approaches has also revealed mechanisms for substrate recognition and at least five defined import pathways for precursor proteins. In this review, we discuss the discovery, recently solved structures, molecular function, and regulation of the TOM complex and its constituents, along with the implications these advances have for alleviating human diseases.

Keywords: TOM complex; Tom40; mitochondrial outer membrane; neurodegeneration; protein import pathways; transmembrane β-barrels.

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Conflict of interest statement

Conflict of interest The authors declare that they no conflict of interest with the contents of this article.

Figures

Figure 1
Figure 1
Structural organization of the TOM–CC.A, components of the TOM–CC and TOM complex (additionally containing one or two copies each of the Tom20 and Tom70 receptors) are shown as cartoon representations. B and C, ribbon diagrams of the Saccharomyces (B) (PDB ID: 6UCU, 6UCV) and human (C) (PDB ID: 7CK6) TOM–CC structures deduced with cryo-EM (26, 27, 28, 29). Pore diameters at the longest and shortest edges are shown in Tom40a and Tom40b, respectively. Both dimeric and tetrameric forms of the TOM–CC have been reported. Tetrameric TOM–CC in (C) was generated using coordinates for the dimer. Tom40 dimerization is facilitated by anchoring interactions of the Tom22 helix and sandwiched detergent/lipid molecules. Tom5, Tom6, and Tom7 associate along the three other faces of Tom40. Tetramerization of the TOM–CC in both ScTOM (B) and HsTOM (C) occurs through Tom6 and stabilized by Tom5–Tom22 interaction in ScTOM. Note how similarities in structure and organization are conserved in both ScTOM and HsTOM. HsTOM, Homo sapiens TOM; ScTOM, Saccharomyces cerevisiae TOM; TOM, translocase of the outer mitochondrial membrane; TOM–CC, TOM core complex.
Figure 2
Figure 2
β-Barrel switching mechanism for SAM-catalyzed assembly of the yeast TOM–CC. Recent cryo-EM structures (56) of the assembly intermediates support a β-barrel switch mechanism for SAM-mediated Tom40 folding. The SAM complex (comprised of Sam35-bound Sam50a and Sam37-bound Sam50b) serves as the chaperone complex for an incoming nascent β-barrel polypeptide. Interaction of the small Tim holdases carrying the nascent Tom40 precursor with the IMS face of Sam50a triggers the dissociation of Sam50b (step I), which initiates the folding of Tom40 in the membrane (step II), leading to formation of the SAM–Tom40 hybrid barrel (Intermediate I) (step III). Sam37 stabilizes the membrane-inserted Tom40 β-barrel by binding at its cytosolic face. The association of Tom5 and Tom6 to the hybrid barrel results in the formation of Intermediate II (step IV). Next, the binding of Tom7 and Tom22 (step V) triggers the dissociation of Tom40/5/6 and its release into the membrane (step Va). The subsequent dimerization of Tom40/5/6/7/22 gives rise to the TOM–CC (step VI). Mdm10 assists Tom40 release, by associating with Sam50a (step Vb). Binding of Sam50b to the hybrid Mdm10–Sam50a/35/37 complex allows reformation of the SAM complex, restoring the chaperone function of the SAM complex (8, 80). The cryo-EM structures (56) also reveal how Tom7 facilitates the dissociation of Tom40/5/6 from the SAM complex (see Fig. 3). Mdm, mitochondrial distribution and morphology; SAM, sorting and assembly machinery; TOM, translocase of the outer mitochondrial membrane; TOM–CC, TOM core complex; IMS, intermembrane space.
Figure 3
Figure 3
Formation of TOM–CC from the SAM–Tom40 hybrid barrel. Tom40 of the SAM–Tom40 hybrid barrel (left; Intermediate I; PDB ID: 7E4H) associates directly with and binds Tom5 and Tom6 through electrostatic interactions, giving rise to Intermediate II (middle; SAM–Tom40/5/6; PDB ID: 7E4I). Tom5/6 binding additionally stabilizes Tom40, while Sam35 stabilizes the elliptical Sam50 barrel during this process (56). Cryo-EM structures of the Tom40–SAM complex reveal a common binding site for both Sam50 and Tom7 on the Tom40 β-barrel (right; purple oval; Tom7 coordinates from PDB ID: 6UCU superimposed on PDB ID: 7E4I), indicating that formation of Tom40–Sam50 and Tom40–Tom7 structures is mutually exclusive. Therefore, the association of Tom7 is anticipated to trigger the release of Tom40/5/6 from the SAM–Tom40 hybrid assembly. Tom7 therefore plays a vital role in the dissociation of Intermediate II (56). SAM, sorting and assembly machinery; TOM, translocase of the outer mitochondrial membrane; TOM–CC, TOM core complex.
Figure 4
Figure 4
Targeting signal sequences for preprotein import by the TOM complex. Proteins imported across the OMM, with various mitochondrial subcompartments as their destination, possess cleavable or noncleavable sequences and can be charged or hydrophobic. The import is orchestrated by a specific TOM complex receptor, and the molecular pathway used by the preprotein within the Tom40 channel is dictated by the polarity of the targeting sequence. Preprotein pathways (A; orange) and (B; magenta), destined for the IMM and matrix respectively, carry an N-terminal cleavable signal sequence, recognized by Tom20. Tom22 interacts with TIM23 of the IMM for coordinated preprotein handover between both OMM and IMM import machineries. TIM23 triggers lateral release of the processed polypeptide in the IMM (A) or completes the import into the matrix (B), after processing by mitochondrial processing peptidases (MPP). IMS proteins with a Cys-rich internal hydrophobic signal sequence (pathway C; brown) are imported in a Tom22-depleted dimeric Tom40 (50). The preprotein is handed over to the IMM-anchored MIA machinery, which catalyzes disulfide bond formation and release of these polypeptides in the IMS. (D; green) OMM β-barrels with a noncleavable C-terminal internal β-signal sequence are recognized first by Tom70 followed by Tom22, for import through Tom40. Tim9–Tim10 holdases handover the unfolded polypeptide to the SAM complex for assembly in the OMM. Carrier proteins (E; blue) are imported first through Tom70–Tom40, carried by small Tims to TIM22, for their import and release in the IMM. (F; purple) Transmembrane helices of the OMM with a noncleavable signal anchor sequence are imported by MIM directly into the OMM, with assistance only from Tom70. Figure inspired from (2, 9, 187). IMM, inner mitochondrial membrane; IMS, intermembrane space; MIA, mitochondrial intermembrane space assembly; MIM, mitochondrial import machinery; OMM, outer mitochondrial membrane; SAM, sorting and assembly machinery; TIM, translocase of the inner mitochondrial membrane; TOM, translocase of the outer mitochondrial membrane.
Figure 5
Figure 5
TOM–TIM interaction for protein import.A, presequence-containing proteins targeted to the mitochondrial matrix require coordinated transfer from the TOM to the TIM23 complex. First, Tom20 recognizes the positively charged presequence and transfers it to Tom22 (step 1). The latter directs the polypeptide to the negatively charged lumen of Tom40 for transport across the OMM through favorable electrostatic interactions (step 2). At the IMS face, the presequence is recognized and bound by the extramembranous domain of Tim50, for transfer to the TIM23 complex (steps 3–4). MPP cleaves the presequence, releasing the mature folded protein in the mitochondrial matrix. The presequence handover from TOM to TIM23 is coordinated by Tom22 and Tim50 in the OMM and IMM, respectively. The matrix face of TIM23 also contains Tim44 and the ATP-dependent presequence translocase-associated motor (PAM). Figure inspired from (68). B, import of multipass transmembrane helical carrier proteins of the IMM is executed by the TIM22 complex. Tom70 recognizes the signal sequence and Tom40 imports it across the OMM (steps 1–2). At the IMS face, small Tim9–Tim10 chaperones capture and retain the polypeptide in its unfolded state (14). The hybrid Tim9–Tim10–Tim12 complex that is formed next, hands over the polypeptide to the TIM22 complex, which successfully inserts and folds the carrier protein into the IMM (steps 3–4). Figure inspired from (63). Both the TOM–TIM import processes are driven by the potential across the IMM and are ATP-independent. IMM, inner mitochondrial membrane; IMS, intermembrane space; MPP, mitochondrial processing peptidase; OMM, outer mitochondrial membrane; TIM, translocase of the inner mitochondrial membrane; TOM, translocase of the outer mitochondrial membrane.
Figure 6
Figure 6
Dynamic interconversion of the TOM complex for selective substrate import. Photo-activated crosslinking revealed a dynamic interconversion of the dimeric and trimeric states of the TOM–CC for import of Cys-rich proteins into the IMS and precursors through Tim50–TIM23, respectively. Mitochondrial porin binds and sequesters Tom22, triggering dissociation of the trimeric state. Phosphorylation of Tom6 favors the trimeric assembly. Figure inspired from (50). IMS, intermembrane space; TIM, translocase of the inner mitochondrial membrane; TOM, translocase of the outer mitochondrial membrane; TOM–CC, TOM core complex.
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