Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex

The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures.     

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.     

Functions of the small proteins in the TOM complex of Neurospora crasssa

The TOM (translocase of the outer mitochondrial membrane) complex of the outer mitochondrial membrane is required for the import of proteins into the organelle. The core TOM complex contains five proteins, including three small components Tom7, Tom6, and Tom5. We have created single and double mutants of all combinations of the three small Tom proteins of Neurospora crassa. Analysis of the mutants revealed that Tom6 plays a major role in TOM complex stability, whereas Tom7 has a lesser role. Mutants lacking both Tom6 and Tom7 have an extremely labile TOM complex and are the only class of mutant to exhibit an altered growth phenotype. Although single mutants lacking N. crassa Tom5 have no apparent TOM complex abnormalities, studies of double mutants lacking Tom5 suggest that it also has a minor role in maintaining TOM complex stability. Our inability to isolate triple mutants supports the idea that the three proteins have overlapping functions. Mitochondria lacking either Tom6 or Tom7 are differentially affected in their ability to import different precursor proteins into the organelle, suggesting that they may play roles in the sorting of proteins to different mitochondrial subcompartments. Newly imported Tom40 was readily assembled into the TOM complex in mitochondria lacking any of the small Tom proteins.     

The TOM core complex: the general protein import pore of the outer membrane of mitochondria

Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.     

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.     

Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.     

Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane

Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space. Unlike studies in fungi, we found that human Tom7 assembles into an approximately 120-kDa import intermediate in HeLa cell mitochondria. To detect subunits within this complex, we employed a novel supershift analysis whereby mitochondria containing newly imported Tom7 were incubated with antibodies specific for individual TOM components prior to separation by blue native electrophoresis. We found that the 120-kDa complex contains Tom40 and lacks receptor components. This intermediate can be chased to the stable approximately 380-kDa mammalian TOM complex that additionally contains Tom22. Overexpression of Tom22 in HeLa cells results in the rapid assembly of Tom7 into the 380-kDa complex indicating that Tom22 is rate-limiting for TOM complex formation. These results indicate that the levels of Tom22 within mitochondria dictate the assembly of TOM complexes and hence may regulate its biogenesis.     

The transmembrane segment of Tom20 is recognized by Mim1 for docking to the mitochondrial TOM complex

Mitochondria cannot be made de novo. Mitochondrial biogenesis requires that up to 1000 proteins are imported into mitochondria, and the protein import pathway relies on hetero-oligomeric translocase complexes in both the inner and outer mitochondrial membranes. The translocase in the outer membrane, the TOM complex, is composed of a core complex formed from the β-barrel channel Tom40 and additional subunits each with single, α-helical transmembrane segments. How α-helical transmembrane segments might be assembled onto a transmembrane β-barrel in the context of a membrane environment is a question of fundamental importance. The master receptor subunit of the TOM complex, Tom20, recognizes the targeting sequence on incoming mitochondrial precursor proteins, binds these protein ligands, and then transfers them to the core complex for translocation across the outer membrane. Here we show that the transmembrane segment of Tom20 contains critical residues essential for docking the Tom20 receptor into its correct environment within the TOM complex. This crucial docking reaction is catalyzed by the unique assembly factor Mim1/Tom13. Mutations in the transmembrane segment that destabilize Tom20, or deletion of Mim1, prevent Tom20 from functioning as a receptor for protein import into mitochondria.     

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

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.     

Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors

The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a approximately 400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.     

The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.     

Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.     

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.     

Identification of Tom5 and Tom6 in the preprotein translocase complex of human mitochondrial outer membrane

The fungal preprotein translocase of the mitochondrial outer membrane (TOM complex) comprises import receptors Tom70, Tom20, and Tom22, import channel Tom40, and small Tom proteins Tom5, Tom6, and Tom7, which regulate TOM complex assembly. These components are conserved in mammals; unlike the other components, however, Tom5 and Tom6 remain unidentified in mammals. We immuno-isolated the TOM complex from HeLa cells expressing hTom22-FLAG and identified the human counterparts of Tom5 and Tom6, together with the other components including Tom7. These small Tom proteins are associated with Tom40 in the TOM complex. Knockdown of Tom7, but not Tom5 and Tom6, strongly compromised stability of the TOM complex. Conversely, knockdown of hTom40 decreased the level of all small Tom proteins. Matrix import of preprotein was affected by double knockdown of any combination of small Tom proteins. These results indicate that human small Tom proteins maintain the structural integrity of the TOM complex.     

Crystal structure of yeast mitochondrial outer membrane translocon member Tom70p

A majority of the proteins targeted to the mitochondria are transported through the translocase of the outer membrane (TOM) complex. Tom70 is a major surface receptor for mitochondrial protein precursors in the TOM complex. To investigate how Tom70 receives the mitochondrial protein precursors, we have determined the crystal structure of yeast Tom70p to 3.0 A. Tom70p forms a homodimer in the crystal. Each subunit consists primarily of tetratricopeptide repeat (TPR) motifs, which are organized into a right-handed superhelix. The TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70, whereas the C-terminal domain of Tom70p contains a large pocket that may be the binding site for mitochondrial precursors. The crystal structure of Tom70p provides insights into the mechanisms of precursor transport across the mitochondrion's outer membrane.     

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.     

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins. The import of cytochrome c is not affected by removal of the receptor Tom20 or Tom70. Moreover, neither the transfer protein Tom5 nor the assembly factors Tom6 and Tom7 are needed for import of cytochrome c. When the general import pore (GIP)-protein Tom40 is blocked, the import of cytochrome c is moderately affected. Mitochondria lacking the central receptor and organizing protein Tom22 contain greatly reduced levels of cytochrome c. We conclude that up to two components of the TOM complex, Tom22 and possibly the GIP, are involved in the biogenesis of cytochrome c.     

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.     

Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane: intermembrane space components are involved in an early stage of the assembly pathway

Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the beta-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of beta-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space.     

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.