A Highlights from MBoC Selection: Assembly of the Mitochondrial Protein Import Channel: Role of Tom5 in Two-Stage Interaction of Tom40 with the SAM Complex

The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central β-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.     

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.     

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.     

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.     

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.     

An Inception Report on the TOM Complex of the Amoeba <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis>, a Simple Model Protozoan in Mitochondria Studies

It is suggested that in the course of the TOM complex evolution at least two lineages have appeared: the animal–fungal and green plant ones. The latter involves also the TOM complexes of algae and protozoans. The amoeba Acanthamoeba castellanii is a free-living nonphotosynthetic soil protozoan, whose mitochondria share many bioenergetic properties with mitochondria of plants, animals and fungi. Here, we report that a protein complex, identified electrophysiologically as the A. castellanii TOM complex, contains a homologue of yeast/animal Tom70. Further, molecular weight of the complex (about 500 kDa) also points to A. castellanii evolutionary relation with fungi and animal. Thus, the data indicates that the TOM complex of A. castellanii is not a typical example of the protozoan TOM complex.     

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.     

Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria

Transport of polypeptides across membranes is a general and essential cellular process utilised by molecular machines. At least one component of these complexes contains a domain composed of three tetratricopeptide repeat (3-TPR) motifs. We have focussed on the receptor Toc64 to elucidate the evolved functional specifications of its 3-TPR domain. Toc64 is a component of the Toc core complex and functionally replaces Tom70 at the outer membrane of mitochondria in plants. Its 3-TPR domain recognises the conserved C-terminus of precursor-bound chaperones. We built homology models of the 3-TPR domain of chloroplastic Toc64 from different species and of the mitochondrial isoform from Arabidopsis. Guided by modelling, we identified residues essential for functional discrimination of the differently located isoforms to be located almost exclusively on the convex surface of the 3-TPR domain. The only exception is at568Ser/ps557Met, which is positioned in the ligand-binding groove. The functional implications of the homology models are discussed. Figure Motion contained within the 2nd eigenvector of the Calpha covariance matrix of the 3-TPR domain of atToc64-V indicated by a porcupine plot Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The TOM complex is involved in the release of superoxide anion from mitochondria

Available data indicate that superoxide anion (O₂^•− ) is released from mitochondria, but apart from VDAC (voltage dependent anion channel), the proteins involved in its transport across the mitochondrial outer membrane still remain elusive. Using mitochondria of the yeast Saccharomyces cerevisiae mutant depleted of VDAC (Δpor1 mutant) and the isogenic wild type, we studied the role of the TOM complex (translocase of the outer membrane) in the efflux of O₂^•− from the mitochondria. We found that blocking the TOM complex with the fusion protein pb₂-DHFR decreased O₂^•− release, particularly in the case of Δpor1 mitochondria. We also observed that the effect of the TOM complex blockage on O₂^•− release from mitochondria coincided with the levels of O₂^•− release as well as with levels of Tom40 expression in the mitochondria. Thus, we conclude that the TOM complex participates in O₂^•− release from mitochondria.     

Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.     

Mdm10 as a dynamic constituent of the TOB/SAM complex directs coordinated assembly of Tom40

The mitochondrial outer membrane contains two protein translocators: the TOM40 and TOB/SAM complexes. Mdm10 is distributed in the TOB complex for β‐barrel protein assembly and in the MMM1 complex for tethering of the endoplasmic reticulum and mitochondria. Here, we establish a system in which the Mdm10 level in the TOB complex—but not in the MMM1 complex—is altered to analyse its part in β‐barrel protein assembly. A decrease in the Mdm10 level results in accumulation of in vitro imported Tom40, which is a β‐barrel protein, at the level of the TOB complex. An increase in the Mdm10 level inhibits association not only of Tom40 but also of other β‐barrel proteins with the TOB complex. These results show that Mdm10 regulates the timing of release of unassembled Tom40 from the TOB complex, to facilitate its coordinated assembly into the TOM40 complex.     

Presequence Recognition by the Tom40 Channel Contributes to Precursor Translocation into the Mitochondrial Matrix

More than 70% of mitochondrial proteins utilize N-terminal presequences as targeting signals. Presequence interactions with redundant cytosolic receptor domains of the translocase of the outer mitochondrial membrane (TOM) are well established. However, after the presequence enters the protein-conducting Tom40 channel, the recognition events that occur at the trans side leading up to the engagement of the presequence with inner membrane-bound receptors are less well defined. Using a photoaffinity-labeling approach with modified presequence peptides, we identified Tom40 as a presequence interactor of the TOM complex. Utilizing mass spectrometry, we mapped Tom40''s presequence-interacting regions to both sides of the β-barrel. Analysis of a phosphorylation site within one of the presequence-interacting regions revealed altered translocation kinetics along the presequence pathway. Our analyses assess the relation between the identified presequence-binding region of Tom40 and the intermembrane space domain of Tom22. The identified presequence-interacting region of Tom40 is capable of functioning independently of the established trans-acting TOM presequence-binding domain during matrix import.     

The mitochondrial TOM complex modulates bax-induced apoptosis in Drosophila

Bax is a pro-apoptotic member of the Bcl-2 family proteins involved in the release of apoptogenic factors from mitochondria to the cytosol. Recently, it has been shown both in mammals and yeast that Bax insertion in the mitochondrial outer membrane involves at least two distinct mechanisms, one of which uses the TOM complex. Here, we show that in Drosophila, heterozygous loss of function mutations of Tom22 or Tom70, two receptors of the TOM complex, attenuates bax-induced phenotypes in vivo. These results argue that the TOM complex may be used as a mitochondrial Bax receptor in Drosophila.     

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.     

Endofin recruits TOM1 to endosomes

Endofin is an endosomal protein implicated in regulating membrane trafficking. It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule. To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system. Three clones that encode TOM1 were recovered. TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family. Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown. Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1. Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1. Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2). The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody. This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin. It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses. In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1. A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxyl-terminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking.     

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the β-barrel–specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a β-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane α-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of α-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to β-barrel proteins but also includes the majority of α-helical Tom proteins.     

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.     

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

A large majority of the 1000–1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K_D of 360 ± 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.     

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.