OMA1 clears traffic jam in TOM tunnel in mammals.

Using an engineered mitochondrial clogger, Krakowczyk et al. (https://doi.org/10.1083/jcb.202306051) identified the OMA1 protease as a critical component that eliminates import failure at the TOM translocase in mammalian cells, providing a novel quality control mechanism that is distinct from those described in yeast.

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.

A first-in-class TIMM44 blocker inhibits bladder cancer cell growth.

Mitochondria play a multifaceted role in supporting bladder cancer progression. Translocase of inner mitochondrial membrane 44 (TIMM44) is essential for maintaining function and integrity of mitochondria. We here tested the potential effect of MB-10 (MitoBloCK-10), a first-in-class TIMM44 blocker, against bladder cancer cells. TIMM44 mRNA and protein expression is significantly elevated in both human bladder cancer tissues and cells. In both patient-derived primary bladder cancer cells and immortalized (T24) cell line, MB-10 exerted potent anti-cancer activity and inhibited cell viability, proliferation and motility. The TIMM44 blocker induced apoptosis and cell cycle arrest in bladder cancer cells, but failed to provoke cytotoxicity in primary bladder epithelial cells. MB-10 disrupted mitochondrial functions in bladder cancer cells, causing mitochondrial depolarization, oxidative stress and ATP reduction. Whereas exogenously-added ATP and the antioxidant N-Acetyl Cysteine mitigated MB-10-induced cytotoxicity of bladder cancer cells. Genetic depletion of TIMM44 through CRISPR-Cas9 method also induced robust anti-bladder cancer cell activity and MB-10 had no effect in TIMM44-depleted cancer cells. Contrarily, ectopic overexpression of TIMM44 using a lentiviral construct augmented proliferation and motility of primary bladder cancer cells. TIMM44 is important for Akt-mammalian target of rapamycin (mTOR) activation. In primary bladder cancer cells, Akt-S6K1 phosphorylation was decreased by MB-10 treatment or TIMM44 depletion, but enhanced after ectopic TIMM44 overexpression. In vivo, intraperitoneal injection of MB-10 impeded bladder cancer xenograft growth in nude mice. Oxidative stress, ATP reduction, Akt-S6K1 inhibition and apoptosis were detected in MB-10-treated xenograft tissues. Moreover, genetic depletion of TIMM44 also arrested bladder cancer xenograft growth in nude mice, leading to oxidative stress, ATP reduction and Akt-S6K1 inhibition in xenograft tissues. Together, targeting overexpressed TIMM44 by MB-10 significantly inhibits bladder cancer cell growth in vitro and in vivo.

Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.

Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early-onset Parkinson's disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin (Ub) ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane forming a high-molecular-weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates Ub, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 N-terminal-C-terminal extension module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery.

The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.

Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.

New insights into the role of mitochondrial metabolic dysregulation and immune infiltration in septic cardiomyopathy by integrated bioinformatics analysis and experimental validation.

BACKGROUND: Septic cardiomyopathy (SCM), a common cardiovascular comorbidity of sepsis, has emerged among the leading causes of death in patients with sepsis. SCM's pathogenesis is strongly affected by mitochondrial metabolic dysregulation and immune infiltration disorder. However, the specific mechanisms and their intricate interactions in SCM remain unclear. This study employed bioinformatics analysis and drug discovery approaches to identify the regulatory molecules, distinct functions, and underlying interactions of mitochondrial metabolism and immune microenvironment, along with potential interventional strategies in SCM. METHODS: GSE79962, GSE171546, and GSE167363 datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and module genes were identified using Limma and Weighted Correlation Network Analysis (WGCNA), followed by functional enrichment analysis. Machine learning algorithms, including support vector machine-recursive feature elimination (SVM-RFE), least absolute shrinkage and selection operator (LASSO) regression, and random forest, were used to screen mitochondria-related hub genes for early diagnosis of SCM. Subsequently, a nomogram was developed based on six hub genes. The immunological landscape was evaluated by single-sample gene set enrichment analysis (ssGSEA). We also explored the expression pattern of hub genes and distribution of mitochondria/inflammation-related pathways in UMAP plots of single-cell dataset. Potential drugs were explored using the Drug Signatures Database (DSigDB). In vivo and in vitro experiments were performed to validate the pathogenetic mechanism of SCM and the therapeutic efficacy of candidate drugs. RESULTS: Six hub mitochondria-related DEGs [MitoDEGs; translocase of inner mitochondrial membrane domain-containing 1 (TIMMDC1), mitochondrial ribosomal protein S31 (MRPS31), F-box only protein 7 (FBXO7), phosphatidylglycerophosphate synthase 1 (PGS1), LYR motif containing 7 (LYRM7), and mitochondrial chaperone BCS1 (BCS1L)] were identified. The diagnostic nomogram model based on the six hub genes demonstrated high reliability and validity in both the training and validation sets. The immunological microenvironment differed between SCM and control groups. The Spearman correlation analysis revealed that hub MitoDEGs were significantly associated with the infiltration of immune cells. Upregulated hub genes showed remarkably high expression in the naive/memory B cell, CD14+ monocyte, and plasma cell subgroup, evidenced by the feature plot. The distribution of mitochondria/inflammation-related pathways varied across subgroups among control and SCM individuals. Metformin was predicted to be the most promising drug with the highest combined score. Its efficacy in restoring mitochondrial function and suppressing inflammatory responses has also been validated. CONCLUSIONS: This study presents a comprehensive mitochondrial metabolism and immune infiltration landscape in SCM, providing a potential novel direction for the pathogenesis and medical intervention of SCM.

The E3 ligase HUWE1 increases the sensitivity of CRC to oxaliplatin through TOMM20 degradation.

Continuous administration of oxaliplatin, the most widely used first-line chemotherapy drug for colorectal cancer (CRC), eventually leads to drug resistance. Increasing the sensitivity of CRC cells to oxaliplatin is a key strategy to overcome this issue. Impairment of mitochondrial function is a pivotal mechanism determining the sensitivity of CRC to oxaliplatin. We discovered an inverse correlation between Translocase of Outer Mitochondrial Membrane 20 (TOMM20) and oxaliplatin sensitivity as well as an inverse relationship between TOMM20 and HECT, UBA, and WWE domain containing E3 ligase 1 (HUWE1) expression in CRC. For the first time, we demonstrated that HUWE1 ubiquitinates TOMM20 directly and also regulates TOMM20 degradation via the PARKIN-mediated pathway. Furthermore, we showed that overexpression of HUWE1 in CRC cells has a negative effect on mitochondrial function, including the generation of ATP and maintenance of mitochondrial membrane potential, leading to increased production of ROS and apoptosis. This effect was amplified when cells were treated simultaneously with oxaliplatin. Our study conclusively shows that TOMM20 is a novel target of HUWE1. Our findings indicate that HUWE1 plays a critical role in regulating oxaliplatin sensitivity by degrading TOMM20 and inducing mitochondrial damage in CRC.

Mitochondrial entry gate as regulatory hub.

Mitochondria import 1000-1300 different precursor proteins from the cytosol. The main mitochondrial entry gate is formed by the translocase of the outer membrane (TOM complex). Molecular coupling and modification of TOM subunits control and modulate protein import in response to cellular signaling. The TOM complex functions as regulatory hub to integrate mitochondrial protein biogenesis and quality control into the cellular proteostasis network.

NADH improves AIF dimerization and inhibits apoptosis in iPSCs-derived neurons from patients with auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.

Mitochondrial Translocase TOMM22 Is Overexpressed in Pancreatic Cancer and Promotes Aggressive Growth by Modulating Mitochondrial Protein Import and Function.

Pancreatic cancer has the worst prognosis among all cancers, underscoring the need for improved management strategies. Dysregulated mitochondrial function is a common feature in several malignancies, including pancreatic cancer. Although mitochondria have their own genome, most mitochondrial proteins are nuclear-encoded and imported by a multi-subunit translocase of the outer mitochondrial membrane (TOMM). TOMM22 is the central receptor of the TOMM complex and plays a role in complex assembly. Pathobiologic roles of TOMM subunits remain largely unexplored. Here we report that TOMM22 protein/mRNA is overexpressed in pancreatic cancer and inversely correlated with disease outcomes. TOMM22 silencing decreased, while its forced overexpression promoted the growth and malignant potential of the pancreatic cancer cells. Increased import of several mitochondrial proteins, including those associated with mitochondrial respiration, was observed upon TOMM22 overexpression which was associated with increased RCI activity, NAD+/NADH ratio, oxygen consumption rate, membrane potential, and ATP production. Inhibition of RCI activity decreased ATP levels and suppressed pancreatic cancer cell growth and malignant behavior confirming that increased TOMM22 expression mediated the phenotypic changes via its modulation of mitochondrial protein import and functions. Altogether, these results suggest that TOMM22 overexpression plays a significant role in pancreatic cancer pathobiology by altering mitochondrial protein import and functions. IMPLICATIONS: TOMM22 bears potential for early diagnostic/prognostic biomarker development and therapeutic targeting for better management of patients with pancreatic cancer.

Tom70-regulated mitochondrial biogenesis via TFAM improves hypoxia-induced dysfunction of pulmonary vascular endothelial cells and alleviates hypoxic pulmonary hypertension.

BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a common type of pulmonary hypertension and characterized by pulmonary vascular remodeling and constriction. A large number of studies have shown that pulmonary vascular endothelial cells (PVECs) dysfunction plays an important role in the initiation and development stages of HPH, but the mechanism of PVECs dysfunction after hypoxia remains unclear. In this study, we explored the exact mechanism of PVECs dysfunction after hypoxia. METHODS: In vitro, we used primary cultured PVECs hypoxia model to mimic HPH injury. We detected the expressions of mitochondrial biogenesis markers, mitochondrial transcription factor A (TFAM) level inside mitochondria, mitochondrial quantity and function, and the components expressions of translocase of outer mitochondrial membrane (TOM) at 24 h after hypoxia. To explore the effects of Tom70 on mitochondrial biogenesis and functions of PVECs after hypoxia, Tom70 overexpression adenovirus was constructed, and the expressions of mitochondrial biogenesis markers, TFAM level inside mitochondria, mitochondrial quantity and function, and the functions of PVECs were detected. And in vivo, we used cre-dependent overexpression adenovirus of Tom70 in the Cdh5-CreERT2 mouse model of HPH to verify the role of upregulating PVECs Tom70 in improving HPH. RESULTS: Hypoxia obviously increased the expressions of mitochondrial biogenesis markers for PGC-1α, NRF-1 and TFAM, but reduced the content of TFAM in mitochondria and the quantity and functions of mitochondria. In addition, only Tom70 expression among the TOM components was significantly decreased after hypoxia, and up-regulation of Tom70 significantly increased the content of TFAM in mitochondria of PVECs by transporting TFAM into mitochondria after hypoxia, enhanced the quantity and functions of mitochondria, improved the functions of PVECs, and ultimately alleviated HPH. CONCLUSION: The findings of present study demonstrated that hypoxia induced the decreased expression of Tom70 in PVECs, reduced the mitochondrial biogenesis-associated TFAM protein transporting into mitochondria, inhibited mitochondrial biogenesis, caused PVECs injury, and prompted the formation of HPH. However, up-regulation of Tom70 abolished the hypoxia-induced injurious effects on PVECs and alleviated HPH.

Genotypic Effects of the TOMM40'523 Variant and APOE on Longitudinal Cognitive Change over 4 Years: The TOMMORROW Study.

BACKGROUND: The 523 poly-T length polymorphism (rs10524523) in TOMM40 has been reported to influence longitudinal cognitive test performance within APOE ε3/3 carriers. The results from prior studies are inconsistent. It is also unclear whether specific APOE and TOMM40 genotypes contribute to heterogeneity in longitudinal cognitive performance during the preclinical stages of AD. OBJECTIVES: To determine the effects of these genes on longitudinal cognitive change in early preclinical stages of AD, we used the clinical trial data from the recently concluded TOMMORROW study to examine the effects of APOE and TOMM40 genotypes on neuropsychological test performance. DESIGN: A phase 3, double-blind, placebo-controlled, randomized clinical trial. SETTING: Academic affiliated and private research clinics in Australia, Germany, Switzerland, the UK, and the USA. PARTICIPANTS: Cognitively normal older adults aged 65 to 83. INTERVENTION: Pioglitazone tablet. MEASUREMENTS: Participants from the TOMMORROW trial were stratified based on APOE genotype (APOE ε3/3, APOE ε3/4, APOE ε4/4). APOE ε3/3 carriers were further stratified by TOMM40'523 genotype. The final analysis dataset consists of 1,330 APOE ε3/3 carriers and 7,001 visits. Linear mixed models were used to compare the rates of decline in cognition across APOE groups and the APOE ε3/3 carriers with different TOMM40'523 genotypes. RESULTS: APOE ε3/4 and APOE ε4/4 genotypes compared with the APOE ε3/3 genotype were associated with worse performance on measures of global cognition, episodic memory, and expressive language. Further, over the four years of observation, the APOE ε3/3 carriers with the TOMM40'523-S/S genotype showed better global cognition and accelerated rates of cognitive decline on tests of global cognition, executive function, and attentional processing compared to APOE ε3/3 carriers with TOMM40'523-S/VL and VL/VL genotypes and compared to the APOE ε3/4 and APOE ε4/4 carriers. CONCLUSIONS: We suggest that both APOE and TOMM40 genotypes may independently contribute to cognitive heterogeneity in the pre-MCI stages of AD. Controlling for this genetic variability will be important in clinical trials designed to slow the rate of cognitive decline and/or prevent symptom onset in preclinical AD.

Alzheimer's disease cortical morphological phenotypes are associated with TOMM40'523-APOE haplotypes.

Both the APOE ε4 and TOMM40 rs10524523 ("523") genes have been associated with risk for Alzheimer's disease (AD) and neuroimaging biomarkers of AD. No studies have investigated the relationship of TOMM40'523-APOE ε4 on the structural complexity of the brain in AD individuals. We quantified brain morphology and multiple cortical attributes in individuals with mild cognitive impairment (MCI) and AD, then tested whether APOE ε4 or TOMM40 poly-T genotypes were related to AD morphological biomarkers in cognitively unimpaired (CU) and MCI/AD individuals. We identified several AD-specific phenotypes in brain morphology and found that TOMM40 poly-T short alleles are associated with early, AD-specific brain morphological differences in healthy aging. We observed decreased cortical thickness, sulcal depth, and fractal dimension in CU individuals with the poly-T short alleles. Moreover, in MCI/AD participants, the APOE ε4 (TOMM40 L) individuals had a higher rate of gene-related morphological markers indicative of AD. Our data suggest that TOMM40'523 is associated with early brain structure variations in the precuneus, temporal, and limbic cortices.

TOMM40 '523 Genotype Distinguishes Patterns of Cognitive Improvement for Executive Function in APOEɛ3 Homozygotes.

BACKGROUND: TOMM40 '523 has been associated with cognitive performance and risk for developing Alzheimer's disease independent of the effect of APOE genotype. Few studies have considered the longitudinal effect of this genotype on change in cognition over time. OBJECTIVE: Our objective was to evaluate the relationship between TOMM40 genotype status and change in cognitive performance in the TOMMORROW study, which was designed to prospectively evaluate an algorithm that includes TOMM40 '523 for genetic risk for conversion to mild cognitive impairment. METHODS: We used latent growth curve models to estimate the effect of TOMM40 allele carrier (short, very long) status on the intercept and slope of change in cognitive performance in four broad cognitive domains (attention, memory, executive function, and language) and a combined overall cognitive score over 30 months. RESULTS: TOMM40 very long allele carriers had significantly lower baseline performance for the combined overall cognitive function score (B = -0.088, p = 0.034) and for the executive function domain score (B = -0.143, p = 0.013). Slopes for TOMM40 very long carriers had significantly greater increases over time for the executive function domain score only. In sensitivity analyses, the results for executive function were observed in participants who remained clinically stable, but not in those who progressed clinically over the study duration. CONCLUSIONS: Our results add to the growing body of evidence that TOMM40, in the absence of APOEɛ4, may contribute to cognitive changes with aging and dementia and support the view that mitochondrial function is an important contributor to Alzheimer's disease risk.

Two domains of Tim50 coordinate translocation of proteins across the two mitochondrial membranes.

Hundreds of mitochondrial proteins with N-terminal presequences are translocated across the outer and inner mitochondrial membranes via the TOM and TIM23 complexes, respectively. How translocation of proteins across two mitochondrial membranes is coordinated is largely unknown. Here, we show that the two domains of Tim50 in the intermembrane space, named core and PBD, both have essential roles in this process. Building upon the surprising observation that the two domains of Tim50 can complement each other in trans, we establish that the core domain contains the main presequence-binding site and serves as the main recruitment point to the TIM23 complex. On the other hand, the PBD plays, directly or indirectly, a critical role in cooperation of the TOM and TIM23 complexes and supports the receptor function of Tim50. Thus, the two domains of Tim50 both have essential but distinct roles and together coordinate translocation of proteins across two mitochondrial membranes.

Molecular pathway of mitochondrial preprotein import through the TOM-TIM23 supercomplex.

Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. The mechanisms through which proteins are translocated via the TOM and TIM23 complexes remain unclear. Here we report the assembly of the active TOM-TIM23 supercomplex of Saccharomyces cerevisiae with translocating polypeptide substrates. Electron cryo-microscopy analyses reveal that the polypeptide substrates pass the TOM complex through the center of a Tom40 subunit, interacting with a glutamine-rich region. Structural and biochemical analyses show that the TIM23 complex contains a heterotrimer of the subunits Tim23, Tim17 and Mgr2. The polypeptide substrates are shielded from lipids by Mgr2 and Tim17, which creates a translocation pathway characterized by a negatively charged entrance and a central hydrophobic region. These findings reveal an unexpected pre-sequence pathway through the TOM-TIM23 supercomplex spanning the double membranes of mitochondria.

Central role of Tim17 in mitochondrial presequence protein translocation.

The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria1,2. About 60% of more than 1,000 different mitochondrial proteins are synthesized with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices3,4. TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views, including regulatory and coupling functions, have been reported on the essential TIM23 subunit Tim17 (refs. 5-7). Here we mapped the interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins during translocation in the native membrane environment. We show that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane.

Mitochondrial microproteins: critical regulators of protein import, energy production, stress response pathways, and programmed cell death.

Mitochondria rely upon the coordination of protein import, protein translation, and proper functioning of oxidative phosphorylation (OXPHOS) complexes I-V to sustain the activities of life for an organism. Each process is dependent upon the function of profoundly large protein complexes found in the mitochondria [translocase of the outer mitochondrial membrane (TOMM) complex, translocase of the inner mitochondrial membrane (TIMM) complex, OXPHOS complexes, mitoribosomes]. These massive protein complexes, in some instances more than one megadalton, are built up from numerous protein subunits of varying sizes, including many proteins that are ≤100-150 amino acids. However, these small proteins, termed microproteins, not only act as cogs in large molecular machines but also have important steps in inhibiting or promoting the intrinsic pathway of apoptosis, coordinate responses to cellular stress, and even act as hormones. This review focuses on microproteins that occupy the mitochondria and are critical for its function. Although the microprotein field is relatively new, researchers have long recognized the existence of these mitochondrial proteins as critical components of virtually all aspects of mitochondrial biology. Thus, recent studies estimating that hundreds of new microproteins of unknown function exist and are missing from current genome annotations suggests that the mitochondrial "microproteome" is a rich area for future biological investigation.

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.