The Outer Mitochondrial Membrane Complex Research Articles

Conserved quality control mechanisms of mitochondrial protein import.

Mitochondria carry out essential functions for the cell, including energy production, various biosynthesis pathways, formation of co-factors and cellular signalling in apoptosis and inflammation. The functionality of mitochondria requires the import of about 900-1300 proteins from the cytosol in baker's yeast Saccharomyces cerevisiae and human cells, respectively. The vast majority of these proteins pass the outer membrane in a largely unfolded state through the translocase of the outer mitochondrial membrane (TOM) complex. Subsequently, specific protein translocases sort the precursor proteins into the outer and inner membranes, the intermembrane space and matrix. Premature folding of mitochondrial precursor proteins, defects in the mitochondrial protein translocases or a reduction of the membrane potential across the inner mitochondrial membrane can cause stalling of precursors at the protein import apparatus. Consequently, the translocon is clogged and non-imported precursor proteins accumulate in the cell, which in turn leads to proteotoxic stress and eventually cell death. To prevent such stress situations, quality control mechanisms remove non-imported precursor proteins from the TOM channel. The highly conserved ubiquitin-proteasome system of the cytosol plays a critical role in this process. Thus, the surveillance of protein import via the TOM complex involves the coordinated activity of mitochondria-localized and cytosolic proteins to prevent proteotoxic stress in the cell.

A converged ubiquitin-proteasome pathway for the degradation of TOC and TOM tail-anchored receptors.

In plants, thousands of nucleus-encoded proteins translated in the cytosol are sorted to chloroplasts and mitochondria by binding to specific receptors of the TOC (translocon on the outer chloroplast membrane) and the TOM (translocon on the outer mitochondrial membrane) complexes for import into those organelles. The degradation pathways for these receptors are unclear. Here, we discovered a converged ubiquitin-proteasome pathway for the degradation of Arabidopsis thaliana TOC and TOM tail-anchored receptors. The receptors are ubiquitinated by E3 ligase(s) and pulled from the outer membranes by the AAA+ adenosine triphosphatase CDC48, after which a previously uncharacterized cytosolic protein, transmembrane domain (TMD)-binding protein for tail-anchored outer membrane proteins (TTOP), binds to the exposed TMDs at the C termini of the receptors and CDC48, and delivers these complexes to the 26S proteasome.

Defective mitochondrial import as a challenge for cellular protein homeostasis.

Mitochondria are organelles indispensable for the correct functioning of eukaryotic cells. Their significance for cellular homeostasis is manifested by the existence of complex quality control pathways that monitor organellar fitness. Mitochondrial biogenesis relies on the efficient import of mitochondrial precursor proteins, a large majority of which are encoded by nuclear DNA and synthesized in the cytosol. This creates a demand for highly specialized import routes that comprise cytosolic factors and organellar translocases. The passage of newly encoded mitochondrial precursor proteins through the cytosol to the translocase of the outer mitochondrial membrane (TOM) is under tight surveillance. As a result of mitochondrial import defects, mitochondrial precursor proteins accumulate in the cytosol or clog the TOM complex, which in turn stimulates cellular stress responses to minimize the consequences of these challenges. These responses are critical for maintaining protein homeostasis under conditions of mitochondrial stress. The present review summarizes recent advances in the field of mitochondrial protein import quality control and discusses the role of this quality control within the network of cellular mechanisms that maintain the cellular homeostasis of proteins.

Mechanism of PINK1 activation by autophosphorylation and insights into assembly on the TOM complex

Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

Structural snapshot of the mitochondrial protein import gate.

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for most mitochondrial proteins. The TOM complex is a multisubunit membrane protein complex consisting of a β-barrel protein Tom40 and six α-helical transmembrane (TM) proteins, receptor subunits Tom20, Tom22, and Tom70, and regulatory subunits Tom5, Tom6, and Tom7. Although nearly 30years have passed since the main components of the TOM complex were identified and characterized, the structural details of the TOM complex remained poorly understood until recently. Thanks to the rapid development of the cryoelectron microscopy (EM) technology, high-resolution structures of the yeast TOM complex have become available. The identified structures showed a symmetric dimer containing five different subunits including Tom22. Biochemical and mutational analyses based on the TOM complex structure revealed the presence of different translocation paths within the Tom40 import channel for different classes of translocating precursor proteins. Previous studies including our cross-linking analyses indicated that the TOM complex in intact mitochondria is present as a mixture of the trimeric complex containing Tom22. Furthermore, the dimeric complex lacking Tom22, and the trimer and dimer may handle different sets of mitochondrial precursor proteins for translocation across the outer membrane. In this Structural Snapshot, we will discuss possible rearrangement of the subunit interactions upon dynamic conversion of the TOM complex between the different subunit assembly states, the Tom22-containing core dimer and trimer.

Atomic structure of human TOM core complex

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for mitochondrial precursor proteins synthesized on cytosolic ribosomes. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the dimeric human TOM core complex (TOM-CC). Two Tom40 β-barrel proteins, connected by two Tom22 receptor subunits and one phospholipid, form the protein-conducting channels. The small Tom proteins Tom5, Tom6, and Tom7 surround the channel and have notable configurations. The distinct electrostatic features of the complex, including the pronounced negative interior and the positive regions at the periphery and center of the dimer on the intermembrane space (IMS) side, provide insight into the preprotein translocation mechanism. Further, two dimeric TOM complexes may associate to form tetramer in the shape of a parallelogram, offering a potential explanation into the unusual structural features of Tom subunits and a new perspective of viewing the import of mitochondrial proteins.

Translocase of the Outer Mitochondrial Membrane 40 Is Required for Mitochondrial Biogenesis and Embryo Development in Arabidopsis.

In eukaryotes, mitochondrion is an essential organelle which is surrounded by a double membrane system, including the outer membrane, intermembrane space and the inner membrane. The translocase of the outer mitochondrial membrane (TOM) complex has attracted enormous interest for its role in importing the preprotein from the cytoplasm into the mitochondrion. However, little is understood about the potential biological function of the TOM complex in Arabidopsis. The aim of the present study was to investigate how AtTOM40, a gene encoding the core subunit of the TOM complex, works in Arabidopsis. As a result, we found that lack of AtTOM40 disturbed embryo development and its pattern formation after the globular embryo stage, and finally caused albino ovules and seed abortion at the ratio of a quarter in the homozygous tom40 plants. Further investigation demonstrated that AtTOM40 is wildly expressed in different tissues, especially in cotyledons primordium during Arabidopsis embryogenesis. Moreover, we confirmed that the encoded protein AtTOM40 is localized in mitochondrion, and the observation of the ultrastructure revealed that mitochondrion biogenesis was impaired in tom40-1 embryo cells. Quantitative real-time PCR was utilized to determine the expression of genes encoding outer mitochondrial membrane proteins in the homozygous tom40-1 mutant embryos, including the genes known to be involved in import, assembly and transport of mitochondrial proteins, and the results demonstrated that most of the gene expressions were abnormal. Similarly, the expression of genes relevant to embryo development and pattern formation, such as SAM (shoot apical meristem), cotyledon, vascular primordium and hypophysis, was also affected in homozygous tom40-1 mutant embryos. Taken together, we draw the conclusion that the AtTOM40 gene is essential for the normal structure of the mitochondrion, and participates in early embryo development and pattern formation through maintaining the biogenesis of mitochondria. The findings of this study may provide new insight into the biological function of the TOM40 subunit in higher plants.

TOM7 silencing exacerbates focal cerebral ischemia injury in rat by targeting PINK1/Beclin1-mediated autophagy

Activated autophagy has been intensively observed in cerebrovascular diseases, including focal cerebral ischemia injury, but its molecular mechanisms remain unclear. TOM7, which is a component of the protein translocase of the outer mitochondrial membrane (TOM) complex, may modulate assembly of the TOM complex. However, an understanding of how TOM7 affects cerebral ischemia injury is limited. In this study, we demonstrate that the expression of TOM7 is up-regulated after a photothrombotic cerebral ischemic model in rats, peaking at 3 days. In addition, TOM7 knockdown may aggravate cerebral ischemic injury and inhibit autophagy after ischemic stroke. Mechanically, TOM7 may regulate autophagy through the PINK1/Beclin1 pathway after cerebral ischemia injury. These results demonstrate that TOM7 silencing may aggravate cerebral ischemia injury through inhibiting PINK1/Beclin1 pathway- mediated autophagy.

The Import of Proteins into the Mitochondrion of Toxoplasma gondii

Outside of well characterized model eukaryotes, relatively little is known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular parasites that cause major diseases in humans and animals and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbor a mitochondrion that is essential for parasite survival and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbors homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for parasite survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for parasite survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan parasites.

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.

Role of membrane contact sites in protein import into mitochondria.

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.

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.

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import

The preprotein translocase of the outer mitochondrial membrane (TOM) functions as the main entry gate for the import of nuclear-encoded proteins into mitochondria. The major subunits of the TOM complex are the three receptors Tom20, Tom22, and Tom70 and the central channel-forming protein Tom40. Cytosolic kinases have been shown to regulate the biogenesis and activity of the Tom receptors. Casein kinase 2 stimulates the biogenesis of Tom22 and Tom20, whereas protein kinase A (PKA) impairs the receptor function of Tom70. Here we report that PKA exerts an inhibitory effect on the biogenesis of the β-barrel protein Tom40. Tom40 is synthesized as precursor on cytosolic ribosomes and subsequently imported into mitochondria. We show that PKA phosphorylates the precursor of Tom40. The phosphorylated Tom40 precursor is impaired in import into mitochondria, whereas the nonphosphorylated precursor is efficiently imported. We conclude that PKA plays a dual role in the regulation of the TOM complex. Phosphorylation by PKA not only impairs the receptor activity of Tom70, but it also inhibits the biogenesis of the channel protein Tom40.

Recognition of Mitochondrial Targeting Sequences by the Import Receptors Tom20 and Tom22

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.

Assembly of the Mitochondrial Protein Import Channel

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.

Targeting and Insertion of the Cholesterol-Binding Translocator Protein into the Outer Mitochondrial Membrane

Translocator protein (18 kDa, TSPO), previously known as the peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import and steroid production. We reconstituted the mitochondrial targeting and insertion of TSPO into the OMM to analyze the signals and mechanisms required for this process. Initial studies indicated the formation of a mitochondrial 66 kDa complex through Blue Native-PAGE analysis. The formation of this complex was found to be dependent on the presence of ATP and the cytosolic chaperone Hsp90. Through mutational analysis we identified two areas necessary for TSPO targeting, import, and function: amino acids 103-108 (Schellman motif), which provide the necessary structural orientation for import, and the cholesterol-binding C-terminus required for insertion. Although the translocase of the outer mitochondrial membrane (TOM) complex proteins Tom22 and Tom40 were present in the OMM, the TOM complex did not interact with TSPO. In search of proteins involved in TSPO import, we analyzed complexes known to interact with TSPO by mass spectrometry. Formation of the 66 kDa complex was found to be dependent on an identified protein, Metaxin 1, for formation and TSPO import. The level of import of TSPO into steroidogenic cell mitochondria was increased following treatment of the cells with cAMP. These findings suggest that the initial targeting of TSPO to mitochondria is dependent upon the presence of cytosolic chaperones interacting with the import receptor Tom70. The C-terminus plays an important role in targeting TSPO to mitochondria, whereas its import into the OMM is dependent upon the presence of the Schellman motif. Final integration of TSPO into the OMM occurs via its interaction with Metaxin 1. Import of TSPO into steroidogenic cell mitochondria is regulated by cAMP.

The Protein Import Channel in the Outer Mitosomal Membrane of Giardia intestinalis

The identification of mitosomes in Giardia generated significant debate on the evolutionary origin of these organelles, whether they were highly reduced mitochondria or the product of a unique endosymbiotic event in an amitochondrial organism. As the protein import pathway is a defining characteristic of mitochondria, we sought to discover a TOM (translocase in the outer mitochondrial membrane) complex in Giardia. A Hidden Markov model search of the Giardia genome identified a Tom40 homologous sequence (GiTom40), where Tom40 is the protein translocation channel of the TOM complex. The GiTom40 protein is located in the membrane of mitosomes in a approximately 200-kDa TOM complex. As Tom40 was derived in the development of mitochondria to serve as the protein import channel in the outer membrane, its presence in Giardia evidences the mitochondrial ancestry of mitosomes.

Interaction of Tim23 with Tim50 Is Essential for Protein Translocation by the Mitochondrial TIM23 Complex

The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.

Cryo-Electron Microscopy Structure of a Yeast Mitochondrial Preprotein Translocase

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for proteins imported into mitochondria. We determined the structure of the native, unstained ∼ 550-kDa core–Tom20 complex from Saccharomyces cerevisiae by cryo-electron microscopy at 18-Å resolution. The complex is triangular, measuring 145 Å on edge, and has near-3-fold symmetry. Its bulk is made up of three globular ∼ 50-Å domains. Three elliptical pores on the c-face merge into one central ∼ 70-Å cavity with a cage-like assembly on the opposite t-face. Nitrilotriacetic acid–gold labeling indicates that three Tom22 subunits in the TOM complex are located at the perimeter of the complex near the interface of the globular domains. We assign Tom22, which controls complex assembly, to three peripheral protrusions on the c-face, while the Tom20 subunit is tentatively assigned to the central protrusion on this surface. Based on our three-dimensional map, we propose a model of transient interactions and functional dynamics of the TOM assembly.