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Mitochondria import the vast majority of their proteins via dedicated protein machineries. The translocase of the outer membrane (TOM complex) forms the main entry site for precursor proteins that are produced on cytosolic ribosomes. Subsequently, different protein sorting machineries transfer the incoming preproteins to the mitochondrial outer and inner membranes, the intermembrane space, and the matrix. In this review, we highlight the recently discovered role of porin, also termed voltage-dependent anion channel (VDAC), in mitochondrial protein biogenesis. Porin forms the major channel for metabolites and ions in the outer membrane of mitochondria. Two different functions of porin in protein translocation have been reported. First, it controls the formation of the TOM complex by modulating the integration of the central receptor Tom22 into the mature translocase. Second, porin promotes the transport of carrier proteins toward the carrier translocase (TIM22 complex), which inserts these preproteins into the inner membrane. Therefore, porin acts as a coupling factor to spatially coordinate outer and inner membrane transport steps. Thus, porin links metabolite transport to protein import, which are both essential for mitochondrial function and biogenesis.

The evolution of mitochondrial protein import and the systems that mediate it marks the boundary between the endosymbiotic ancestor of mitochondria and a true organelle that is under the control of the nucleus. Protein import has been studied in great detail in Saccharomyces cerevisiae. More recently, it has also been extensively investigated in the parasitic protozoan Trypanosoma brucei, making it arguably the second best studied system. A comparative analysis of the protein import complexes of yeast and trypanosomes is provided. Together with data from other systems, this allows to reconstruct the ancestral features of import complexes that were present in the last eukaryotic common ancestor (LECA) and to identify which subunits were added later in evolution. How these data can be translated into plausible scenarios is discussed, providing insights into the evolution of (i) outer membrane protein import receptors, (ii) proteins involved in biogenesis of α-helically anchored outer membrane proteins, and (iii) of the intermembrane space import and assembly system. Finally, it is shown that the unusual presequence-associated import motor of trypanosomes suggests a scenario of how the two ancestral inner membrane protein translocases present in LECA evolved into the single bifunctional one found in extant trypanosomes.

The mitochondrial AAA ATPase Msp1 is well known for extraction of mislocalized tail-anchored ER proteins from the mitochondrial outer membrane. Here, we analyzed the extraction of precursors blocking the import pore in the outer membrane. We demonstrate strong genetic interactions of Msp1 and the proteasome with components of the TOM complex, the main translocase in the outer membrane. Msp1 and the proteasome both contribute to the removal of arrested precursor proteins that specifically accumulate in these mutants. The proteasome activity is essential for the removal as proteasome inhibitors block extraction. Furthermore, the proteasomal subunit Rpn10 copurified with Msp1. The human Msp1 homologue has been implicated in neurodegenerative diseases, and we show that the lack of the Caenorhabditis elegans Msp1 homologue triggers an import stress response in the worm, which indicates a conserved role in metazoa. In summary, our results suggest a role of Msp1 as an adaptor for the proteasome that drives the extraction of arrested and mislocalized proteins at the mitochondrial outer membrane.

Mitochondria harbor in their outer membrane (OM) proteins of different topologies. These proteins are encoded by the nuclear DNA, translated on cytosolic ribosomes and inserted into their target organelle by sophisticated protein import machineries. Recently, considerable insights have been accumulated on the insertion pathways of proteins into the mitochondrial OM. In contrast, little is known regarding the early cytosolic stages of their biogenesis. It is generally presumed that chaperones associate with these proteins following their synthesis in the cytosol, thereby keeping them in an import-competent conformation and preventing their aggregation and/or mis-folding and degradation. In this review, we outline the current knowledge about the biogenesis of different mitochondrial OM proteins with various topologies, and highlight the recent findings regarding their import pathways starting from early cytosolic events until their recognition on the mitochondrial surface that lead to their final insertion into the mitochondrial OM.

TOM70 is a member of the TOM complex that transports cytosolic proteins into mitochondria. Here, we identified two compound heterozygous variants in TOMM70 [c.794C>T (p.T265M) and c.1745C>T (p.A582V)] from a patient with severe anemia, lactic acidosis, and developmental delay. Patient-derived immortalized lymphocytes showed decreased TOM70 expression, oligomerized TOM70 complex, and TOM 20/22/40 complex compared with expression in control lymphocytes. Functional analysis revealed that patient-derived cells exhibited multi-oxidative phosphorylation system (OXPHOS) complex defects, with complex IV being primarily affected. As a result, patient-derived cells grew slower in galactose medium and generated less ATP and more extracellular lactic acid than did control cells. In vitro cell model compensatory experiments confirmed the pathogenicity of TOMM70 variants since only wild-type TOM70, but not mutant TOM70, could restore the complex IV defect and TOM70 expression in TOM70 knockdown U2OS cells. Altogether, we report the first case of mitochondrial disease-causing mutations in TOMM70 and demonstrate that TOM70 is essential for multi-OXPHOS assembly. Mutational screening of TOMM70 should be employed to identify mitochondrial disease-causing gene mutations in the future.

Mitochondrial biogenesis has two major steps: the transcriptional activation of nuclear genome-encoded mitochondrial proteins and the import of nascent mitochondrial proteins that are synthesized in the cytosol. These nascent mitochondrial proteins are aggregation-prone and can cause cytosolic proteostasis stress. The transcription factor-dependent transcriptional regulations and the TOM-TIM complex-dependent import of nascent mitochondrial proteins have been extensively studied. Yet, little is known regarding how these two steps of mitochondrial biogenesis coordinate with each other to avoid the cytosolic accumulation of these aggregation-prone nascent mitochondrial proteins. Here we show that in budding yeast, Tom70, a conserved receptor of the TOM complex, moonlights to regulate the transcriptional activity of mitochondrial proteins. Tom70’s transcription regulatory role is conserved in Drosophila. The dual roles of Tom70 in both transcription/biogenesis and import of mitochondrial proteins allow the cells to accomplish mitochondrial biogenesis without compromising cytosolic proteostasis. The age-related reduction of Tom70, caused by reduced biogenesis and increased degradation of Tom70, is associated with the loss of mitochondrial membrane potential, mtDNA, and mitochondrial proteins. While loss of Tom70 accelerates aging and age-related mitochondrial defects, overexpressing TOM70 delays these mitochondrial dysfunctions and extends the replicative lifespan. Our results reveal unexpected roles of Tom70 in mitochondrial biogenesis and aging.

A role for reversible phosphorylation in regulation of mitochondrial proteins has been neglected for a long time. Particularly, the import machineries that mediate influx of more than 1000 different precursor proteins into the organelle were considered as predominantly constitutively active entities. Only recently, a combination of advanced phosphoproteomic approaches and Phos-tag technology enabled the discovery of several phosphorylation sites at the translocase of the outer membrane TOM and the identification of cellular signalling cascades that allow dynamic adaptation of the protein influx into mitochondria upon changing cellular demands. Here, we present a protocol that allows biochemical and semi-quantitative profiling of intra-mitochondrial protein phosphorylation. We exemplify this with the pyruvate dehydrogenase complex (PDH), which serves as a central metabolic switch in energy metabolism that is based on reversible phosphorylation. Phos-tag technology allows rapid monitoring of the metabolic state via simultaneous detection of phosphorylated and non-phosphorylated species of the PDH core component Pda1. Our protocol can be applied for several further intra-organellar proteins like respiratory chain complexes or protein translocases of the inner membrane. SIGNIFICANCE: Our manuscript describes for the first time how Phos-tag technology can be applied to monitor phosphorylation of intramitochondrial proteins. We exemplify this with the regulation of the pyruvate dehydrogenase complex as central regulatory switch in energy metabolism. We show that our protocol allows a rapid monitoring of the metabolic state of the cell (phosphorylated PDH is inactive while non-phosphorylated PDH is active) and can be applied for rapid profiling of different metabolic conditions as well as for profiling phosphorylation of further intramitochondrial protein (complexes).

Mitochondria are essential organelles for cellular energy production, metabolic homeostasis, calcium homeostasis, cell proliferation, and apoptosis. About 99% of mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and imported into mitochondria by mitochondrial protein import machinery. Mitochondrial protein import systems function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles. Mitochondrial protein import deficiency is linked to various diseases, including cardiovascular disease. In this review, we describe an emerging class of protein or genetic variations of components of the mitochondrial import machinery involved in heart disease. The major protein import pathways, including the presequence pathway (TIM23 pathway), the carrier pathway (TIM22 pathway), and the mitochondrial intermembrane space import and assembly machinery, related translocases, proteinases, and chaperones, are discussed here. This review highlights the importance of mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease.

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.

Mitochondria import about 1000 proteins that are produced as precursors on cytosolic ribosomes. Defects in mitochondrial protein import result in the accumulation of non-imported precursor proteins and proteotoxic stress. The cell is equipped with different quality control mechanisms to monitor protein transport into mitochondria. First, molecular chaperones guide unfolded proteins to mitochondria and deliver non-imported proteins to proteasomal degradation. Second, quality control factors remove translocation stalled precursor proteins from protein translocases. Third, protein translocases monitor protein sorting to mitochondrial subcompartments. Fourth, AAA proteases of the mitochondrial subcompartments remove mislocalized or unassembled proteins. Finally, impaired efficiency of protein transport is an important sensor for mitochondrial dysfunction and causes the induction of cellular stress responses, which could eventually result in the removal of the defective mitochondria by mitophagy. In this review, we summarize our current understanding of quality control mechanisms that govern mitochondrial protein transport.

Mitochondria are essential eukaryotic organelles responsible for primary cellular energy production. Biogenesis, maintenance, and functions of mitochondria require correct assembly of resident proteins and lipids, which require their transport into and within mitochondria. Mitochondrial normal functions also require an exchange of small metabolites between the cytosol and mitochondria, which is primarily mediated by a metabolite channel of the outer membrane (OM) called porin or voltage-dependent anion channel. Here, we describe recently revealed novel roles of porin in the mitochondrial protein and lipid transport. First, porin regulates the formation of the mitochondrial protein import gate in the OM, the translocase of the outer membrane (TOM) complex, and its dynamic exchange between the major form of a trimer and the minor form of a dimer. The TOM complex dimer lacks a core subunit Tom22 and mediates the import of a subset of mitochondrial proteins while the TOM complex trimer facilitates the import of most other mitochondrial proteins. Second, porin interacts with both a translocating inner membrane (IM) protein like a carrier protein accumulated at the small TIM chaperones in the intermembrane space and the TIM22 complex, a downstream translocator in the IM for the carrier protein import. Porin thereby facilitates the efficient transfer of carrier proteins to the IM during their import. Third, porin facilitates the transfer of lipids between the OM and IM and promotes a back-up pathway for the cardiolipin synthesis in mitochondria. Thus, porin has roles more than the metabolite transport in the protein and lipid transport into and within mitochondria, which is likely conserved from yeast to human.

Mitochondrial biogenesis requires the import of a large number of precursor proteins from the cytosol. Although specific membrane-bound preprotein translocases have been characterized in detail, it was assumed that protein transfer from the cytosol to mitochondria mainly involved unselective binding to molecular chaperones. Recent findings suggest an unexpected versatility of protein transfer to mitochondria. Cytosolic factors have been identified that bind to selected subsets of preproteins and guide them to mitochondrial receptors in a post-translational manner. Cotranslational import processes are emerging. Mechanisms for crosstalk between protein targeting to mitochondria and other cell organelles, in particular the endoplasmic reticulum (ER) and peroxisomes, have been uncovered. We discuss how a network of cytosolic machineries and targeting pathways promote and regulate preprotein transfer into mitochondria.

Mitochondrial biogenesis and functions depend on the import of precursor proteins via the 'translocase of the outer membrane' (TOM complex). Defects in protein import lead to an accumulation of mitochondrial precursor proteins that induces a range of cellular stress responses. However, constitutive quality-control mechanisms that clear trapped precursor proteins from the TOM channel under non-stress conditions have remained unknown. Here we report that in Saccharomyces cerevisiae Ubx2, which functions in endoplasmic reticulum-associated degradation, is crucial for this quality-control process. A pool of Ubx2 binds to the TOM complex to recruit the AAA ATPase Cdc48 for removal of arrested precursor proteins from the TOM channel. This mitochondrial protein translocation-associated degradation (mitoTAD) pathway continuously monitors the TOM complex under non-stress conditions to prevent clogging of the TOM channel with precursor proteins. The mitoTAD pathway ensures that mitochondria maintain their full protein-import capacity, and protects cells against proteotoxic stress induced by impaired transport of proteins into mitochondria.

In this report, we summarize recent findings about a role of the outer membrane metabolite channel VDAC/porin in protein import into mitochondria. Mitochondria fulfill key functions for cellular energy metabolism. Their biogenesis involves the import of about 1000 different proteins that are produced as precursors on cytosolic ribosomes. The translocase of the outer membrane (TOM complex) forms the entry gate for mitochondrial precursor proteins. Dedicated protein translocases sort the preproteins into the different mitochondrial subcompartments. While protein transport pathways are analyzed to some detail, only little is known about regulatory mechanisms that fine-tune protein import upon metabolic signaling. Recently, a dual role of the voltage-dependent anion channel (VDAC), also termed porin, in mitochondrial protein biogenesis was reported. First, VDAC/porin promotes as a coupling factor import of carrier proteins into the inner membrane. Second, VDAC/porin regulates the formation of the TOM complex. Thus, the major metabolite channel in the outer membrane VDAC/porin connects protein import to mitochondrial metabolism.

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 MICOS complex mediates formation of the crista junctions in mitochondria. Here we analyzed the mitochondrial import pathways for the six yeast MICOS subunits as a step toward understanding of the assembly mechanisms of the MICOS complex. Mic10, Mic12, Mic26, Mic27, and Mic60 used the presequence pathway to reach the intermembrane space (IMS). In contrast, Mic19 took the TIM40/MIA pathway, through its CHCH domain, to reach the IMS. Unlike canonical TIM40/MIA substrates, presence of the N-terminal unfolded DUF domain impaired the import efficiency of Mic19, yet N-terminal myristoylation of Mic19 circumvented this effect. The myristoyl group of Mic19 binds to Tom20 of the TOM complex as well as the outer membrane, which may lead to “entropy pushing” of the DUF domain followed by the CHCH domain of Mic19 into the import channel, thereby achieving efficient import.

The central role mitochondria play in cellular homeostasis has made its study critical to our understanding of various aspects of human health and disease. Mitochondria rely on the translocase of the outer membrane (TOM) complex for the bulk of mitochondrial protein import. In addition to its role as the major entry point for mitochondrial proteins, the TOM complex serves as an entry pathway for viral proteins. TOM complex subunits also participate in a host of interactions that have been studied extensively for their function in neurodegenerative diseases, cardiovascular diseases, innate immunity, cancer, metabolism, mitophagy and autophagy. Recent advances in our structural understanding of the TOM complex and the protein import machinery of the outer mitochondrial membrane have made structure-based therapeutics targeting outer mitochondrial membrane proteins during mitochondrial dysfunction an exciting prospect. Here, we describe advances in understanding the TOM complex, the interactome of the TOM complex subunits, the implications for the development of therapeutics, and our understanding of the structure/function relationship between components of the TOM complex and mitochondrial homeostasis.

β-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial β-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial β-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.

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 30 years 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.

BackgroundThe uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Distinct fractions of proteins initially bind to the receptor proteins and are subsequently transferred to the pore-forming component for import. The aim of this study was the identification of the decisive elements of this machinery that determine the specific selection of the proteins that should be imported.ResultsWe identified the essential internal targeting signal of the members of the mitochondrial metabolite carrier proteins, the largest protein family of the mitochondria, and we investigated the specific recognition of this signal by the protein import machinery at the mitochondrial outer surface. We found that the outer membrane import receptors facilitated the uptake of these proteins, and we identified the corresponding binding site, marked by cysteine C141 in the receptor protein Tom70. However, in tests both in vivo and in vitro, the import receptors were neither necessary nor sufficient for specific recognition of the targeting signals. Although these signals are unrelated to the amino-terminal presequences that mediate the targeting of other mitochondrial preproteins, they were found to resemble presequences in their strict dependence on a content of positively charged residues as a prerequisite of interactions with the import pore.ConclusionsThe general import pore of the mitochondrial outer membrane appears to represent not only the central channel of protein translocation but also to form the decisive general selectivity filter in the uptake of the newly synthesized mitochondrial proteins.

Outer membrane proteins integrate mitochondria into the cellular environment. They warrant exchange of small molecules like metabolites and ions, transport proteins into mitochondria, form contact sites to other cellular organelles for lipid exchange, constitute a signaling platform for apoptosis and inflammation and mediate organelle fusion and fission. The outer membrane contains two types of integral membrane proteins. Proteins with a transmembrane β-barrel structure and proteins with a single or multiple α-helical membrane spans. All outer membrane proteins are produced on cytosolic ribosomes and imported into the target organelle. Precursors of β-barrel and α-helical proteins are transported into the outer membrane via distinct import routes. The translocase of the outer membrane (TOM complex) transports β-barrel precursors across the outer membrane and the sorting and assembly machinery (SAM complex) inserts them into the target membrane. The mitochondrial import (MIM) complex constitutes the major integration site for α-helical embedded proteins. The import of some MIM-substrates involves TOM receptors, while others are imported in a TOM-independent manner. Remarkably, TOM, SAM and MIM complexes dynamically interact to import a large set of different proteins and to coordinate their assembly into protein complexes. Thus, protein import into the mitochondrial outer membrane involves a dynamic platform of protein translocases.

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and distinct protein quality control (PQC) machineries resulting different protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to sub-mitochondrial compartments, we fused well-described bipartite or matrix-targeting signal sequences to misfolding and aggregation-prone stressor proteins to target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, using transcriptomics and proteomics, we report a comprehensive stress response elicited by two types of targeting signal-fused stressor proteins. Among global responses by mitochondria-targeted stressors by both types of signal sequences, an adaptive response of abrogated mitochondrial respiration and concomitant upregulation of glycolysis is uncovered. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that bipartite signal sequence-fused stressor proteins eliciting stress to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

A new inhibitor of the deubiquitylase USP30, an actionable target relevant to Parkinson’s Disease, is introduced and characterised for parameters related to mitophagy., The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinson’s Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

Given their polyvalent functions, an inherent challenge that mitochondria face is the exposure to mitochondrial import stresses, culminating in their dysfunction. Recently, mitochondrial import of several mitochondrial substrates was shown to be regulated via a 'tug of war' between USP30 and MARCH5, two ubiquitin-related enzymes located at the TOM complex.

Mitochondrial dysregulation is a pivotal hallmark of aging-related disorders. Although there is a considerable understanding of the molecular counteracting responses toward damaged mitochondria, the molecular underpinnings connecting the abnormal aggregation of mitochondrial precursor protein fragments and abrogation of mitochondrial import machinery are far from clear. Recently, proteasomal-dependent degradation was unveiled as a pivotal fine-tuner of TOM machinery-dependent mitochondrial import. Herein, the role of proteasomal-mediated degradation in regulating fidelity of TOM-dependent import is briefly discussed and analyzed. The insights obtained from the characterization of this process may be applied to targeting mitochondrial import dysfunction in some neurodegenerative disorders.

In the past three decades, significant advances have been made in providing the biochemical background of TOM (translocase of the outer mitochondrial membrane)-mediated protein translocation into mitochondria. In the light of recent cryoelectron microscopy-derived structures of TOM isolated from Neurospora crassa and Saccharomyces cerevisiae, the interpretation of biochemical and biophysical studies of TOM-mediated protein transport into mitochondria now rests on a solid basis. In this review, we compare the subnanometer structure of N. crassa TOM core complex with that of yeast. Both structures reveal remarkably well-conserved symmetrical dimers of 10 membrane protein subunits. The structural data also validate predictions of weakly stable regions in the transmembrane β-barrel domains of the protein-conducting subunit Tom40, which signal the existence of β-strands located in interfaces of protein-protein interactions.

Mitochondrial precursor proteins with amino-terminal presequences are imported via the presequence pathway, utilizing the TIM23 complex for inner membrane translocation. Initially, the precursors pass the outer membrane through the TOM complex and are handed over to the TIM23 complex where they are sorted into the inner membrane or translocated into the matrix. This handover process depends on the receptor proteins at the inner membrane, Tim50 and Tim23, which are critical for efficient import. In this review, we summarize key findings that shaped the current concepts of protein translocation along the presequence import pathway, with a particular focus on the precursor handover process from TOM to the TIM23 complex. In addition, we discuss functions of the human TIM23 pathway and the recently uncovered pathogenic mutations in TIM50.

Mitochondria are deeply integrated into crucial functions of eukaryotic cells, including ATP production via oxidative phosphorylation, biosynthesis of iron-sulfur clusters, amino acids, lipids and heme, signaling pathways, and programmed cell death. The import of about 1000 different proteins that are produced as precursors on cytosolic ribosomes is essential for mitochondrial functions and biogenesis. The translocase of the outer mitochondrial membrane (TOM) forms the entry gate for the vast majority of mitochondrial proteins. Research of the last years has uncovered a complicated network of protein translocases and pathways that sort proteins into the mitochondrial subcompartments: outer and inner membranes, intermembrane space, and matrix. The in vitro import of a large number of different precursor proteins into mitochondria has been a pivotal experimental assay to identify these protein-sorting routes. This experimental set-up enables studies on the kinetics of protein transport into isolated mitochondria, on the processing of precursor proteins, and on their assembly into functional protein machineries. In vitro protein import assays are widely used and are indispensable for research on mitochondrial protein biogenesis.

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.

Summary Mitochondria possess elaborate machineries for the import of proteins from the cytosol. Cytosolic factors like Hsp70 chaperones and their co-chaperones, the J-proteins, guide proteins to the mitochondrial surface. The translocase of the mitochondrial outer membrane (TOM) forms the entry gate for preproteins. How the proteins are delivered to mitochondrial preprotein receptors is poorly understood. We identify the cytosolic J-protein Xdj1 as a specific interaction partner of the central receptor Tom22. Tom22 recruits Xdj1 to the mitochondrial surface to promote import of preproteins and assembly of the TOM complex. Additionally, we find that the receptor Tom70 binds a different cytosolic J-protein, Djp1. Our findings suggest that cytosolic J-proteins target distinct TOM receptors and promote the biogenesis of mitochondrial proteins., Graphical Abstract, Highlights • The receptor Tom22 recruits the cytosolic J-protein Xdj1 to mitochondria • Xdj1 delivers preproteins to Tom22 and promotes biogenesis of the TOM complex • The receptor Tom70 recruits a different cytosolic J-protein, Djp1 • Mitochondrial receptors selectively recognize cytosolic J-protein co-chaperones, Opaliński et al. report that mitochondrial protein import receptors selectively recognize J-protein co-chaperones of the cytosol. The co-chaperones bind hydrophobic precursor proteins and assist in transferring them to the receptors of the mitochondrial protein entry gate.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a nuclear and mitochondrial protein that in nuclei and in vitro repairs blocked 3′ DNA termini such as 3′ phosphotyrosine conjugates resulting from stalling of topoisomerase I-DNA intermediates. Its mutation also causes spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1). Because Tdp1 colocalizes with mitochondria following oxidative stress, we hypothesized that Tdp1 repairs mitochondrial DNA (mtDNA) and that mtDNA damage mediates entry of Tdp1 into the mitochondria. To test this, we used S. cerevisiae mutants, cultured mouse and human cells, and a Tdp1 knockout mouse. H2O2- and rotenone-induced cellular and intramitochondrial reactive oxygen species (ROS) activated oxidant-responsive kinases P38 and ERK1, and the translocation of Tdp1 from the nucleus to the mitochondria via the TIM/TOM complex. This translocation occurred independently of mtDNA. Within the mitochondria, Tdp1 interacted with Ligase III and reduced mtDNA mutations. Tdp1-deficient tissues had impaired mitochondrial respiration and decreased viability. These observations suggest that Tdp1 maintains mtDNA integrity and support the hypothesis that mitochondrial dysfunction contributes to the pathology of SCAN1.

Finally, the architecture of the translocase of the mitochondrial outer membrane (TOM complex) is revealed, after 20 years of anticipation. Two groups have now determined the near-atomic structures of the TOM complex. These findings improve understanding of the mechanisms by which TOM facilitates the passage of about 1,000 different proteins from the cytosol into the mitochondria.

Mitochondrial biogenesis and functions rely on transport of their resident proteins as well as small molecules/ions across their membranes. The TOM complex functions as a protein entry gate for most mitochondrial proteins and mitochondrial porin facilitates transport of small-molecule metabolites and ions. We recently found a novel role of porin in regulation of the TOM complex assembly, the dynamic exchange between the dimer and trimer, and different substrate specificities of the dimer and trimer. Using distinct assembly forms customized for different client proteins, the TOM complex can handle ~ 1000 different mitochondrial protein for their import into mitochondria.

A variable-length poly-T variant in intron 6 of the TOMM40 gene, rs10524523, is associated with risk and age-of-onset of sporadic (late-onset) Alzheimer's disease. In Caucasians, the three predominant alleles at this locus are Short (S), Long (L) or Very long (VL). On an APOE e3/3 background, the S/VL and VL/VL genotypes are more protective than S/S. The '523 poly-T has regulatory properties, in that the VL poly-T results in higher expression than the S poly-T in luciferase expression systems. The aim of the current work was to identify effects on cellular bioenergetics of increased TOM40 protein expression. MitoTracker Green fluorescence and autophagic vesicle staining was the same in control and over-expressing cells, but TOM40 over-expression was associated with increased expression of TOM20, a preprotein receptor of the TOM complex, the mitochondrial chaperone HSPA9, and PDHE1a, and increased activities of the oxidative phosphorylation complexes I and IV and of the TCA member α-ketoglutaric acid dehydrogenase. Consistent with the complex I findings, respiration was more sensitive to inhibition by rotenone in control cells than in the TOM40 over-expressing cells. In the absence of inhibitors, total cellular ATP, the mitochondrial membrane potential, and respiration were elevated in the over-expressing cells. Spare respiratory capacity was greater in the TOM40 over-expressing cells than in the controls. TOM40 over-expression blocked Ab-elicited decreases in the mitochondrial membrane potential, cellular ATP levels, and cellular viability in the control cells. These data suggest elevated expression of TOM40 may be protective of mitochondrial function.

The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network.

Protein transport systems are fundamentally important for maintaining mitochondrial function. Nevertheless, mitochondrial protein translocases such as the kinetoplastid ATOM complex have recently been shown to vary in eukaryotic lineages. Various evolutionary hypotheses have been formulated to explain this diversity. To resolve any contradiction, estimating the primitive state and clarifying changes from that state are necessary. Here, we present more likely primitive models of mitochondrial translocases, specifically the translocase of the outer membrane (TOM) and translocase of the inner membrane (TIM) complexes, using scrutinized phylogenetic profiles. We then analyzed the translocases’ evolution in eukaryotic lineages. Based on those results, we propose a novel evolutionary scenario for diversification of the mitochondrial transport system. Our results indicate that presequence transport machinery was mostly established in the last eukaryotic common ancestor, and that primitive translocases already had a pathway for transporting presequence-containing proteins. Moreover, secondary changes including convergent and migrational gains of a presequence receptor in TOM and TIM complexes, respectively, likely resulted from constrained evolution. The nature of a targeting signal can constrain alteration to the protein transport complex.

Mitochondria have many different functions the most important one of which is oxidative phosphorylation. They originated from an endosymbiotic event between a bacterium and an archaeal host cell. It was the evolution of a protein import system that marks the boundary between the endosymbiotic ancestor of the mitochondrion and a true organelle that is under the control of the nucleus. In present day mitochondria more than 95% of all proteins are imported from the cytosol, in a process mediated by hetero-oligomeric protein complexes in the outer and inner mitochondrial membranes. In this review we compare mitochondrial protein import in the best studied model system yeast and the parasitic protozoan Trypanosoma brucei. The two organisms are phylogenetically only remotely related. Despite the fact that mitochondrial protein import has the same function in both species, only very few subunits of their import machineries are conserved. Moreover, while yeast has two inner membrane protein translocases, one specialized for presequence-containing and one for mitochondrial carrier proteins, T. brucei has a single inner membrane translocase only, that mediates import of both types of substrates. The evolutionary implications of these findings are discussed.

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria following synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. Using cryo-EM, we have obtained high-resolution structures of both apo and presequence-bound core TOM complexes from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry—Tom40, a pore-forming β-barrel with an overall negatively-charged inner surface, and four auxiliary α-helical transmembrane proteins. The structure suggests that presequences for mitochondrial targeting insert into the Tom40 channel mainly by electrostatic and polar interactions. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Our study reveals the molecular organization of the TOM complex and provides new insights about the mechanism of protein translocation into mitochondria.

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria following synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. We have determined high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry, pore-forming β-barrel protein Tom40 and four auxiliary α-helical transmembrane proteins. The pore of each Tom40 has an overall negatively charged inner surface attributed to multiple functionally important acidic patches. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Our study reveals the detailed molecular organization of the TOM complex and provides new insights about the mechanism of protein translocation into mitochondria.

Summary: The mitochondrial outer membrane contains integral proteins with α-helical membrane anchors or a transmembrane β-barrel. The translocase of the outer membrane (TOM) cooperates with the sorting and assembly machinery (SAM) in the import of β-barrel proteins, whereas the mitochondrial import (MIM) complex inserts precursors of multi-spanning α-helical proteins. Single-spanning proteins constitute more than half of the integral outer membrane proteins; however, their biogenesis is poorly understood. We report that the yeast MIM complex promotes the insertion of proteins with N-terminal (signal-anchored) or C-terminal (tail-anchored) membrane anchors. The MIM complex exists in three dynamic populations. MIM interacts with TOM to accept precursor proteins from the receptor Tom70. Free MIM complexes insert single-spanning proteins that are imported in a Tom70-independent manner. Finally, coupling of MIM and SAM promotes early assembly steps of TOM subunits. We conclude that the MIM complex is a major and versatile protein translocase of the mitochondrial outer membrane. : Doan et al. report that the mitochondrial import (MIM) complex constitutes the major import site for single-spanning and multi-spanning outer membrane proteins. MIM exists in three forms and dynamically cooperates with outer membrane protein translocases to import different types of precursor proteins. Keywords: MIM complex, mitochondria, outer membrane, protein assembly, protein sorting, protein translocase, SAM complex, TOM complex

Summary In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1, 2, 3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4, 5, 6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7, 8, 9, 10, 11, 12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes., Highlights • Substrates of mitochondrial TIM22 complex identified by proteomics in S. cerevisiae • Carrier proteins with six membrane spans confirmed as substrates • Pyruvate carrier (MPC) subunits (two or three membrane spans) transported by TIM22 • MPC import dependence on TIM22 is conserved from yeast to human, Mitochondria carrier proteins, which possess six transmembrane spans (TM), are transported by the TIM22 complex. By using a quantitative proteomic approach, Gomkale and Cruz-Zaragoza et al. reveal subunits of the mitochondrial pyruvate carrier (MPC) that possess two or three TMs as unexpected substrates of the carrier pathway.

Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space toward the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the mitochondrial contact site and cristae organizing system (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria, the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane. peerReviewed

Despite the progress in understanding the molecular responses to mitochondrial damage, responses to aberrant accumulation of mitochondrial precursor proteins and mitochondrial import defects remain poorly understood. Recent work (Martensson et al., Nature, 2019) has unveiled a pathway similar to endoplasmic-reticulum-associated degradation (ERAD) in fine-tuning the fidelity of translocase of the outer mitochondrial membrane (TOM) complex-mediated mitochondrial import.

Biogenesis and function of mitochondria depend on the import of about 1000 precursor proteins that are produced on cytosolic ribosomes. The translocase of the outer membrane (TOM) forms the entry gate for most proteins. After passage through the TOM channel, dedicated preprotein translocases sort the precursor proteins into the mitochondrial subcompartments. Many proteins have to be assembled into oligomeric membrane-integrated complexes in order to perform their functions. In this review, we discuss a dual role of mitochondrial preprotein translocases in protein translocation and oligomeric assembly, focusing on the biogenesis of the TOM complex and the respiratory chain. The sorting and assembly machinery (SAM) of the outer mitochondrial membrane forms a dynamic platform for coupling transport and assembly of TOM subunits. The biogenesis of the cytochrome c oxidase of the inner membrane involves a molecular circuit to adjust translation of mitochondrial-encoded core subunits to the availability of nuclear-encoded partner proteins. Thus, mitochondrial protein translocases not only import precursor proteins but can also support their assembly into functional complexes.

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.

Porin is crucial for metabolite flux in mitochondria. In this issue of Molecular Cell, Sakaue et al. (2019) and Ellenrieder et al. (2019) describe an unexpected role for Porin in mitochondrial protein import by regulating the oligomeric state of the major protein import gate, the TOM complex, and the inner membrane insertion of metabolite carriers.

The diverse functions of mitochondria depend on hundreds of different proteins. The vast majority of these proteins is encoded in the nucleus, translated in the cytosol, and must be imported into the organelle. Import was shown to occur after complete synthesis of the protein, with the assistance of cytosolic chaperones that maintain it in an unfolded state and target it to the mitochondrial translocase of the outer membrane (TOM complex). Recent studies, however, identified many mRNAs encoding mitochondrial proteins near the outer membrane of mitochondria. Translation studies suggest that many of these mRNAs are translated locally, presumably allowing cotranslational import into mitochondria. Herein we review these data and discuss its relevance for local protein synthesis. We also suggest alternative roles for mRNA localization to mitochondria. Finally, we suggest future research directions, including revealing the significance of localization to mitochondria physiology and the molecular players that regulate it.

Mitochondria acquire the majority of their proteins from the cytosol in a process that is mediated by intricate multimeric machineries designed to allow proteins to cross and/or to insert themselves into the two mitochondrial membranes. Ongoing studies carried out in yeast over the past few decades have led to the discovery of numerous protein components that constitute several mitochondrial translocases. One of these complexes, the mitochondrial TIM23, is the major translocase for matrix proteins and is the focus of this review. The components of the TIM23 complex are categorized into four functional types. The first type plays the role of receptor for preproteins in the intermembrane space. The second type forms the actual channel that allows proteins to cross the inner mitochondrial membrane. The third species functions as part of the motor that mediates the final steps of import across the inner membrane. Additional components play regulatory roles orchestrating the action of this myriad of subunits. Recent studies provide new insights into the function of the mammalian TIM23 complex and the role that it plays under pathological conditions.