Role of the TOM Complex in Protein Import into Mitochondria: Structural Views.

Mitochondria are central to energy production, metabolism and signaling, and apoptosis. To make new mitochondria from preexisting mitochondria, the cell needs to import mitochondrial proteins from the cytosol into the mitochondria with the aid of translocators in the mitochondrial membranes. The translocase of the outer membrane (TOM) complex, an outer membrane translocator, functions as an entry gate for most mitochondrial proteins. Although high-resolution structures of the receptor subunits of the TOM complex were deposited in the early 2000s, those of entire TOM complexes became available only in 2019. The structural details of these TOM complexes, consisting of the dimer of the ß-barrel import channel Tom40 and four α-helical membrane proteins, revealed the presence of several distinct paths and exits for the translocation of over 1,000 different mitochondrial precursor proteins. High-resolution structures of TOM complexes now open up a new era of studies on the structures, functions, and dynamics of the mitochondrial import system.

Cryo-EM Structure of the TOM Core Complex from Neurospora crassa.

The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the ß-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery.

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.

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

Dynamic Regulation of Mitochondrial Import by the Ubiquitin System.

Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.

Porin Associates with Tom22 to Regulate the Mitochondrial Protein Gate Assembly.

Mitochondria import nearly all of their resident proteins from the cytosol, and the TOM complex functions as their entry gate. The TOM complex undergoes a dynamic conversion between the majority population of a three-channel gateway ("trimer") and the minor population that lacks Tom22 and has only two Tom40 channels ("dimer"). Here, we found that the porin Por1 acts as a sink to bind newly imported Tom22. This Por1 association thereby modulates Tom22 integration into the TOM complex, guaranteeing formation of the functional trimeric TOM complex. Por1 sequestration of Tom22 dissociated from the trimeric TOM complex also enhances the dimeric TOM complex, which is preferable for the import of TIM40/MIA-dependent proteins into mitochondria. Furthermore, Por1 appears to contribute to cell-cycle-dependent variation of the functional trimeric TOM complex by chaperoning monomeric Tom22, which arises from the cell-cycle-controlled variation of phosphorylated Tom6.

Reciprocal Roles of Tom7 and OMA1 during Mitochondrial Import and Activation of PINK1.

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.

TOM-1/tomosyn acts with the UNC-6/netrin receptor UNC-5 to inhibit growth cone protrusion in Caenorhabditis elegans.

In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion.

Two conformations of the Tom20 preprotein receptor in the TOM holo complex.

The TOM complex is the main entry point for precursor proteins (preproteins) into mitochondria. Preproteins containing targeting sequences are recognized by the TOM complex and imported into mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle electron cryomicroscopy at 3.3 Å resolution, showing its interaction with a bound preprotein at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6 to 7 Å resolution. TOM is a transmembrane complex consisting of two ß-barrels, three receptor subunits, and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding preproteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.

From powerhouse to processing plant: conserved roles of mitochondrial outer membrane proteins in tRNA splicing.

The mitochondrial cytoplasmic surface serves as a processing site for numerous RNAs from budding yeast to metazoans. We report that budding yeast mitochondrial outer membrane (MOM) proteins that are subunits of the translocase of the outer mitochondrial membrane (Tom70 and Tom 22) and sorting and assembly machinery (Sam37) are required for efficient pretransfer RNA (pre-tRNA) splicing. Defective pre-tRNA splicing in MOM mutants is due not to loss of respiratory metabolism but instead inefficient targeting/tethering of tRNA splicing endonuclease (SEN) subunits to mitochondria. Schizosaccharomyces pombe SEN subunits also localize to mitochondria, and Tom70 is required for this localization and pre-tRNA splicing. Thus, the role of MOM protein in targeting/tethering SEN subunits to mitochondria has been conserved for >500 million years.

Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors.

Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive. Here we determined the structure of TOM core complex at 2.53 Å and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 Å. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain. Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.

The TOM complex from an evolutionary perspective and the functions of TOMM70.

In humans, up to 1,500 mitochondrial precursor proteins are synthesized at cytosolic ribosomes and must be imported into the organelle. This is not only essential for mitochondrial but also for many cytosolic functions. The majority of mitochondrial precursor proteins are imported over the translocase of the outer membrane (TOM). In recent years, high-resolution structure analyses from different organisms shed light on the composition and arrangement of the TOM complex. Although significant similarities have been found, differences were also observed, which have been favored during evolution and could reflect the manifold functions of TOM with cellular signaling and its response to altered metabolic situations. A key component within these regulatory mechanisms is TOMM70, which is involved in protein import, forms contacts to the ER and the nucleus, but is also involved in cellular defense mechanisms during infections.

New insights into the structure and dynamics of the TOM complex in mitochondria.

To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.

The neural structures of theory of mind are valence-sensitive: evidence from three tDCS studies.

Several cortical structures are involved in theory of mind (ToM), including the dorsolateral prefrontal cortex (dlPFC), the ventromedial prefrontal cortex (vmPFC), and the right temporo- parietal junction (rTPJ). We investigated the role of these regions in mind reading with respect to the valence of mental states. Sixty-five healthy adult participants were recruited and received transcranial direct current stimulation (tDCS) (1.5 mA, 20 min) with one week interval in three separate studies. The stimulation conditions were anodal tDCS over the dlPFC coupled with cathodal tDCS over the vmPFC, reversed stimulation conditions, and sham in the first study, and anodal tDCS over the vmPFC, or dlPFC, and sham stimulation, with an extracranial return electrode in the second and third study. During stimulation, participants underwent the reading mind from eyes/voice tests (RMET or RMVT) in each stimulation condition. Anodal left dlPFC/cathodal right vmPFC stimulation increased the accuracy of negative mental state attributions, anodal rTPJ decreased the accuracy of negative and neutral mental state attributions, and decreased the reaction time of positive mental state attributions. Our results imply that the neural correlates of ToM are valence-sensitive.

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.

SIRT5 induces autophagy and alleviates myocardial infarction via desuccinylation of TOM1.

Myocardial infarction (MI) is a prevalent form of ischemic heart disease, significantly contributing to heart disease-related deaths worldwide. This condition is primarily caused by myocardial ischemic-reperfusion injury (MIRI). Sirtuin 5 (SIRT5) is a desuccinylase known for its ability to reduce protein succinylation. Recent studies have highlighted the potential role of SIRT5 in various human diseases, including MIRI. This study aims to investigate the specific role of SIRT5 in modulating autophagy and cardiomyocyte death in a MIRI model, as well as to identify the downstream protein targets of SIRT5. Initially, we established a hypoxia/reoxygenation (H/R)-induced MIRI cell model to measure SIRT5 expression and assess its functions. Our results indicated that H/R induction led to a downregulation of SIRT5 expression, decreased autophagy, and increased cell death. Notably, overexpression of SIRT5 effectively promoted autophagy and inhibited cell death in the MIRI cell model. Mechanistically, SIRT5 was found to directly interact with the target of myb1 membrane trafficking protein (TOM1) at the K48 site, inducing its desuccinylation and stabilization. Further rescue assays revealed that TOM1 knockdown reversed the changes in autophagy and apoptosis caused by SIRT5 overexpression in the MIRI cell model. In vivo experiments demonstrated that SIRT5 alleviated myocardial injury in MI models. In conclusion, this study uncovers the role of SIRT5-mediated desuccinylation of TOM1 in regulating autophagy-related cell death in MIRI, providing new insights into potential therapeutic strategies for MI.

The Translocase of the Outer Mitochondrial Membrane (TOM40) is required for mitochondrial dynamics and neuronal integrity in Dorsal Root Ganglion Neurons.

Polymorphisms and altered expression of the Translocase of the Outer Mitochondrial Membrane - 40 kD (Tom40) are observed in neurodegenerative disease subjects. We utilized in vitro cultured dorsal root ganglion (DRG) neurons to investigate the association of TOM40 depletion to neurodegeneration, and to unravel the mechanism of neurodegeneration induced by decreased levels of TOM40 protein. We provide evidence that severity of neurodegeneration induced in the TOM40 depleted neurons increases with the increase in the depletion of TOM40 and is exacerbated by an increase in the duration of TOM40 depletion. We also demonstrate that TOM40 depletion causes a surge in neuronal calcium levels, decreases mitochondrial motility, increases mitochondrial fission, and decreases neuronal ATP levels. We observed that alterations in the neuronal calcium homeostasis and mitochondrial dynamics precede BCL-xl and NMNAT1 dependent neurodegenerative pathways in the TOM40 depleted neurons. This data also suggests that manipulation of BCL-xl and NMNAT1 may be of therapeutic value in TOM40 associated neurodegenerative disorders.

Dihydroartemisinin-driven TOM70 inhibition leads to mitochondrial destabilization to induce pyroptosis against lung cancer.

Enhancement of malignant cell immunogenicity to relieve immunosuppression of lung cancer microenvironment is essential in lung cancer treatment. In previous study, we have demonstrated that dihydroartemisinin (DHA), a kind of phytopharmaceutical, is effective in inhibiting lung cancer cells and boosting their immunogenicity, while the initial target of DHA's intracellular action is poorly understood. The present in-depth analysis aims to reveal the influence of DHA on the highly expressed TOM70 in the mitochondrial membrane of lung cancer. The affinity of DHA and TOM70 was analyzed by microscale thermophoresis (MST), pronase stability, and thermal stability. The functions and underlying mechanism were investigated using western blots, qRT-PCR, flow cytometry, and rescue experiments. TOM70 inhibition resulted in mtDNA damage and translocation to the cytoplasm from mitochondria due to the disruption of mitochondrial homeostasis. Further ex and in vivo findings also showed that the cGAS/STING/NLRP3 signaling pathway was activated by mtDNA and thereby malignant cells underwent pyroptosis, leading to enhanced immunogenicity of lung cancer cells in the presence of DHA. Nevertheless, DHA-induced mtDNA translocation and cGAS/STING/NLRP3 mobilization were synchronously attenuated when TOM70 was replenished. Finally, DHA was demonstrated to possess potent anti-lung cancer efficacy in vitro and in vivo. Taken together, these data confirm that TOM70 is an important target for DHA to disturb mitochondria homeostasis, which further activates STING and arouses pyroptosis to strengthen immunogenicity against lung cancer thereupon. The present study provides vital clues for phytomedicine-mediated anti-tumor therapy.

Prefoldin 2 contributes to mitochondrial morphology and function.

BACKGROUND: Prefoldin is an evolutionarily conserved co-chaperone of the tailless complex polypeptide 1 ring complex (TRiC)/chaperonin containing tailless complex 1 (CCT). The prefoldin complex consists of six subunits that are known to transfer newly produced cytoskeletal proteins to TRiC/CCT for folding polypeptides. Prefoldin function was recently linked to the maintenance of protein homeostasis, suggesting a more general function of the co-chaperone during cellular stress conditions. Prefoldin acts in an adenosine triphosphate (ATP)-independent manner, making it a suitable candidate to operate during stress conditions, such as mitochondrial dysfunction. Mitochondrial function depends on the production of mitochondrial proteins in the cytosol. Mechanisms that sustain cytosolic protein homeostasis are vital for the quality control of proteins destined for the organelle and such mechanisms among others include chaperones. RESULTS: We analyzed consequences of the loss of prefoldin subunits on the cell proliferation and survival of Saccharomyces cerevisiae upon exposure to various cellular stress conditions. We found that prefoldin subunits support cell growth under heat stress. Moreover, prefoldin facilitates the growth of cells under respiratory growth conditions. We showed that mitochondrial morphology and abundance of some respiratory chain complexes was supported by the prefoldin 2 (Pfd2/Gim4) subunit. We also found that Pfd2 interacts with Tom70, a receptor of mitochondrial precursor proteins that are targeted into mitochondria. CONCLUSIONS: Our findings link the cytosolic prefoldin complex to mitochondrial function. Loss of the prefoldin complex subunit Pfd2 results in adaptive cellular responses on the proteome level under physiological conditions suggesting a continuous need of Pfd2 for maintenance of cellular homeostasis. Within this framework, Pfd2 might support mitochondrial function directly as part of the cytosolic quality control system of mitochondrial proteins or indirectly as a component of the protein homeostasis network.

The relationship between empathy, theory of mind and facial affect recognition in schizophrenia patients.

Although impairments in social cognition are a core feature in schizophrenia, the relationship between its subcomponents is less clear. Nineteen schizophrenia patients and 20 matched healthy controls were tested for emotion recognition, and for the cognitive and affective subcomponents of empathy and theory of mind (ToM). Patients scored significantly worse than controls on cognitive empathy and both subcomponents of ToM. Group differences disappeared for cognitive empathy and affective ToM when emotion recognition was included as a covariate. Our results indicate that emotion recognition is an important factor involved in the deficits on higher levels of social cognition in schizophrenia.