Biogenesis of Tom40, core component of the TOM complex of mitochondria.

Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH(2) nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH(2)-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.     

Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.     

Assembly of Tom6 and Tom7 into the TOM core complex of Neurospora crassa

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.     

Structural insights into proapoptotic signaling mediated by MTCH2, VDAC2, TOM40 and TOM22

Mitochondrial Outer Membrane (MOM) Permeabilization (MOMP) is a critical step in the intrinsic pathway of apoptosis and is regulated by the Bcl-2 family of proteins. In vitro studies using cardiolipin-containing liposomes as a MOM model have suggested that a mitochondria-specific phospholipid, cardiolipin, is of crucial importance in MOMP. However, recently it has been found that the MOM contains much less cardiolipin than it is required for liposome permeabilization. Shortly thereafter, several MOM proteins, such as VDAC2, MTCH2, TOM22 and TOM40, have been identified as the Bax, Bak and tBid receptors that are indispensable in MOMP, but the underlying mechanisms are elusive. Here, proapoptotic signaling mediated by these MOM receptors was explored in terms of 3D-structures of interacting proteins using computational modeling. The formation under apoptotic conditions of the TOM40/TOM22/tBid protein complex possessing a fairly high binding affinity towards Bax is predicted, suggesting the recruitment of Bax to mitochondria by this complex in apoptotic cells. Our simulations predict the displacement of Bax from the TOM40/TOM22/tBid/Bax complex by another Bax in auto-catalytic manner and explain, in terms of structure, the tBid-mediated displacement of Bak from the VDAC2/Bak complex. Computational modeling revealed high-affinity binding of Bid to MTCH2 suggesting both a quasi-constitutive residence of Bid in MTCH2-bound state in healthy cells and its caspase-8-mediated cleavage there under apoptotic conditions. Overall, our results provide structural details for important stages of apoptotic signaling mediated by MOM receptors and enrich its mechanistic understanding.     

Effect of mutations in Tom40 on stability of the translocase of the outer mitochondrial membrane (TOM) complex, assembly of Tom40, and import of mitochondrial preproteins

Mitochondrial preproteins synthesized in the cytosol are imported through the mitochondrial outer membrane by the translocase of the outer mitochondrial membrane (TOM) complex. Tom40 is the major component of the complex and is essential for cell viability. We generated 21 different mutations in conserved regions of the Neurospora crassa Tom40 protein. The mutant genes were transformed into a tom40 null nucleus maintained in a sheltered heterokaryon, and 17 of the mutant genes gave rise to viable strains. All mutations reduced the efficiency of the altered Tom40 molecules to assemble into the TOM complex. Mitochondria isolated from seven of the mutant strains had defects for importing mitochondrial preproteins. Only one strain had a general import defect for all preproteins examined. Another mutation resulted in defects in the import of a matrix-destined preprotein and an outer membrane beta-barrel protein, but import of the ADP/ATP carrier to the inner membrane was unaffected. Five strains showed deficiencies in the import of beta-barrel proteins. The latter results suggest that the TOM complex distinguishes beta-barrel proteins from other classes of preprotein during import. This supports the idea that the TOM complex plays an active role in the transfer of preproteins to subsequent translocases for insertion into the correct mitochondrial subcompartment.     

Role of Tom5 in maintaining the structural stability of the TOM complex of mitochondria

Transport of nuclear encoded proteins into mitochondria is mediated by multisubunit translocation machineries in the outer and inner membranes of mitochondria. The TOM complex contains receptor and pore components that facilitate the recognition of preproteins and their transfer through the outer membrane. In addition, the complex contains a set of small proteins. Tom7 and Tom6 have been found in Neurospora and yeast, Tom5 has been found so far only in the latter organism. In the present study, we identified Neurospora Tom5 and analyzed its function in comparison to yeast Tom5, which has been proposed to play a role as a receptor-like component. Neurospora Tom5 crosses the outer membrane with its carboxyl terminus facing the intermembrane space like the other small Tom components. The temperature-sensitive growth phenotype of the yeast TOM5 deletion was rescued by overexpression of Neurospora Tom5. On the other hand, Neurospora cells deficient in tom5 did not exhibit any defect in growth. The structural stability of TOM complexes from cells devoid of Tom5 was significantly altered in yeast but not in Neurospora. The efficiency of protein import in Neurospora mitochondria was not affected by deletion of tom5, whereas in yeast it was reduced as compared with wild type. We conclude that the main role of Tom5, rather than being a receptor, is maintaining the structural integrity of the TOM complex.     

Characterization of Neurospora crassa Tom40-deficient mutants and effect of specific mutations on Tom40 assembly

The TOM complex (Translocase of the Outer mitochondrial Membrane) is responsible for the recognition of mitochondrial preproteins synthesized in the cytosol and for their translocation across or into the outer mitochondrial membrane. Tom40 is the major component of the TOM complex and forms the translocation pore. We have created a tom40 mutant of Neurospora crassa and have demonstrated that the gene is essential for the viability of the organism. Mitochondria with reduced levels of Tom40 were deficient for import of mitochondrial preproteins and contained reduced levels of the TOM complex components Tom22 and Tom6, suggesting that the import and/or stability of these proteins is dependent on the presence of Tom40. Mutant Tom40 preproteins were analyzed for their ability to be assembled into the TOM complex. In vitro import assays revealed that conserved regions near the N terminus (residues 51-60) and the C terminus (residues 321-323) of the 349-amino acid protein were required for assembly beyond a 250-kDa intermediate form. Mutant strains expressing Tom40 with residues 51-60 deleted were viable but exhibited growth defects. Slow growing mutants expressing Tom40, where residues 321-323 were changed to Ala residues, were isolated but showed TOM complex defects, whereas strains in which residues 321-323 were deleted could not be isolated. Analysis of the assembly of mutant Tom40 precursors in vitro supported a previous model in which Tom40 precursors progress from the 250-kDa intermediate to a 100-kDa form and then assemble into the 400-kDa TOM complex. Surprisingly, when wild type mitochondria containing Tom40 precursors arrested at the 250-kDa intermediate were treated with sodium carbonate, further assembly of intermediates into the TOM complex occurred, suggesting that disruption of protein-protein interactions may facilitate assembly. Import of wild type Tom40 precursor into mitochondria containing a mutant Tom40 lacking residues 40-48 revealed an alternate assembly pathway and demonstrated that the N-terminal region of pre-existing Tom40 molecules in the TOM complex plays a role in the assembly of incoming Tom40 molecules.     

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins.     

Mim1, a protein required for the assembly of the TOM complex of mitochondria

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into mitochondria. This complex is essential for the formation and maintenance of mitochondria. Here, we report on the role of the integral outer membrane protein, Mim1 (mitochondrial import), in the biogenesis of mitochondria. Depletion of Mim1 abrogates assembly of the TOM complex and results in accumulation of Tom40, the principal constituent of the TOM complex, as a low-molecular-mass species. Like all mitochondrial beta-barrel proteins, the precursor of Tom40 is inserted into the outer membrane by the TOB complex. Mim1 is likely to be required for a step after this TOB-complex-mediated insertion. Mim1 is a constituent of neither the TOM complex nor the TOB complex; rather, it seems to be a subunit of another, as yet unidentified, complex. We conclude that Mim1 has a vital and specific function in the assembly of the TOM complex.     

Dissection of the mitochondrial import and assembly pathway for human Tom40

Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane. By examining the import pathway of human Tom40, we have been able to elucidate additional features in its import. We identify that Hsp90 is involved in delivery of the Tom40 precursor to mitochondria in an ATP-dependent manner. The precursor then forms its first stable intermediate with the outer face of the TOM complex before its membrane integration and assembly. Deletion of an evolutionary conserved region within Tom40 disrupts the TOM complex intermediate and causes it to stall at a new complex in the intermembrane space that we identify to be the mammalian SAM. Unlike its fungal counterparts, the human Tom40 precursor is not found stably arrested at a SAM intermediate. Nevertheless, we show that Tom40 assembly is reduced in mitochondria depleted of human Sam50. These findings are discussed in context with current models from fungal studies.     

Structural insights into the assembly of human translesion polymerase complexes

In addition to DNA repair pathways, cells utilize translesion DNA synthesis (TLS) to bypass DNA lesions during replication. During TLS, Y-family DNA polymerase (Polη, Polκ, Polι and Rev1) inserts specific nucleotide opposite preferred DNA lesions, and then Polζ consisting of two subunits, Rev3 and Rev7, carries out primer extension. Here, we report the complex structures of Rev3-Rev7-Rev1^CTD and Rev3-Rev7-Rev1^CTDPolκ^RIR. These two structures demonstrate that Rev1^CTD contains separate binding sites for Pol- and Rev7. Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1. We also verified through FRET experiment that Rev1, Rev3, Rev7 and Polκ form a stable quaternary complex in vivo, thereby suggesting an efficient switching mechanism where the “inserter” polymerase can be immediately replaced by an “extender” polymerase within the same quaternary complex.     

Tom7 regulates Mdm10-mediated assembly of the mitochondrial import channel protein Tom40

β-barrel membrane proteins in the mitochondrial outer membrane use the TOM40 complex to enter mitochondria and then the TOB/SAM complex to be assembled into the outer membrane. Tom7, a subunit of the TOM40 complex, regulates association of Mdm10 with the TOB complex. Here, we analyzed the role of Tom7 in assembly of β-barrel proteins, including Tom40, a central channel subunit of the TOM40 complex, and porin. Depletion of Tom7 decreased transient accumulation of Tom40 at the level of the TOB complex and retarded assembly of porin in vitro. On the other hand, overexpression of Tom7 resulted in enhanced accumulation of in vitro imported Tom40 in the TOB complex, yet it did not affect the in vitro assembly of porin. Site-specific photocross-linking in vivo revealed that Tom7 directly interacts with Tom40 through its transmembrane segment and with Mdm10. These results collectively show that Tom7 recruits Mdm10, enhancing its association with the MMM1 complex, to regulate timing of the release of Tom40 from the TOB complex for subsequent assembly into the TOM40 complex.     

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.     

The receptor subunit Tom20 is dynamically associated with the TOM complex in mitochondria of human cells

The outer membrane translocase (TOM) is the import channel for nuclear-encoded mitochondrial proteins. The general import pore contains Tom40, Tom22, Tom5, Tom6, and Tom7. Precursor proteins are bound by the (peripheral) receptor proteins Tom20, Tom22, and Tom70 before being imported by the TOM complex. Here we investigated the association of the receptor Tom20 with the TOM complex. Tom20 was found in the TOM complex, but not in a smaller subcomplex. In addition, a subcomplex was found without Tom40 and Tom7 but with Tom20. Using single particle tracking of labeled Tom20 in overexpressing human cells, we show that Tom20 has, on average, higher lateral mobility in the membrane than Tom7/TOM. After ligation of Tom20 with the TOM complex by post-tranlational protein trans-splicing using the traceless, ultrafast cleaved Gp41-1 integrin system, a significant decrease in the mean diffusion coefficient of Tom20 was observed in the resulting Tom20–Tom7 fusion protein. Exposure of Tom20 to high substrate loading also resulted in reduced mobility. Taken together, our data show that the receptor subunit Tom20 interacts dynamically with the TOM core complex. We suggest that the TOM complex containing Tom20 is the active import pore and that Tom20 is associated when substrate is available.     

Structural requirements for the assembly of Norwalk virus-like particles

Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.     

Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity

We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis.     

Identification and functional analysis of human Tom22 for protein import into mitochondria

Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.     

Structural requirements for duodenal permeability of heparin-diamine complexes.

We have compared the physico-chemical behaviors alone and in the presence of a synthetic bilayer membrane, in aqueous solution and the bioavailability after intraduodenal administration to rabbits, of the two heparin diamine salts ITF-300 and ITF-331 with those of the heparin-amine salt ITF-1175. The three salts have similar structures but different characteristics of compounds tend to form aggregates in solution, but at different critical concentrations. The compounds induce fusion of single-walled vesicles of a synthetic peptide lipid into multi-walled lamellae. The minimal concentrations of the compounds required for the formation of such lamellae differ. This behavior in solution explains the differences in absorption in the animal model. This makes it possible to correlate enhanced heparin bioavailability with the structural nature of the diamine counter-ions used to prepare heparin salts.     

Targeting and assembly of rat mitochondrial translocase of outer membrane 22 (TOM22) into the TOM complex

Tom22 is a preprotein receptor and organizer of the mitochondrial outer membrane translocase complex (TOM complex). Rat Tom22 (rTOM22) is a 142-residue protein, embedded in the outer membrane through the internal transmembrane domain (TMD) with 82 N-terminal residues in the cytosol and 41 C-terminal residues in the intermembrane space. We analyzed the signals that target rTOM22 to the mitochondrial outer membrane and assembly into the TOM complex in cultured mammalian cells. Deletions or mutations were systematically introduced into the molecule, and the intracellular localization of the mutant constructs in HeLa cells was examined by confocal microscopy and cell fractionation. Their assembly into the TOM complex was also examined using blue native gel electrophoresis. These experiments revealed three separate structural elements: a cytoplasmic 10-residue segment with an acidic alpha-helical structure located 30 residues upstream of the TMD (the import sequence), TMD with an appropriate hydrophobicity, and a 20-residue C-terminal segment located 22 residues downstream of the TMD (C-tail signal). The import sequence and TMD were both essential for targeting and integration into the TOM complex, whereas the C-tail signal affected the import efficiency. The import sequence combined with foreign TMD functioned as a mitochondrial targeting and anchor signal but failed to integrate the construct into the TOM complex. Thus, the mitochondrial-targeting and TOM integration signal could be discriminated. A yeast two-hybrid assay revealed that the import sequence interacted with two intramolecular elements, the TMD and C-tail signal, and that it also interacted with the import receptor Tom20.