The Outer Mitochondrial Membrane Research Articles

Bi-Allelic Splicing Variant, c.153-2A > C in TOMM7 Is Associated With Leigh Syndrome.

Translocase of the outer mitochondrial membrane (TOMM) complex plays an important role in the transport of proteins from the cytoplasm into the mitochondria. TOMM7, one of the subunits of the TOMM complex, modulates its assembly and stability. Bi-allelic disease-causing variants in TOMM7 (MIM* 607980) have been previously reported in two unrelated families with a diverse phenotype of short stature, lipodystrophy, progeria, developmental delay, hypotonia, and skeletal dysplasia. We report a 4-month-old female child significantly affected with neonatal-onset hypotonia, lactic acidosis, optic atrophy, and neuroimaging findings suggestive of Leigh disease with a novel canonical splice variant, c.153-2A > C in TOMM7 (NM_019059.5). Further work done on cDNA of parents revealed the presence of shorter transcripts secondary to aberrant splicing.

Conserved quality control mechanisms of mitochondrial protein import.

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

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

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

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.

Defective mitochondrial import as a challenge for cellular protein homeostasis.

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

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

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

Structural snapshot of the mitochondrial protein import gate.

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

Atomic structure of human TOM core complex

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

The structure of the TOM core complex in the mitochondrial outer membrane.

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.

TOMM40 polymorphism is associated with resting-state functional MRI results in patients with Alzheimer's disease.

Translocase of outer mitochondrial membrane 40 (TOMM40) encodes translocase of the outer mitochondrial membrane (TOM), which is associated with mitochondrial dysfunction in Alzheimer's disease (AD). TOMM40 rs157581-G has been reported to increase susceptibility to AD. However, the effect of TOMM40 rs157581-G in resting-state functional MRI (rs-fMRI) on AD has not been studied. Therefore, we aimed to investigate the role of TOMM40 rs157581-G on rs-fMRI results in AD patients. Twenty-four AD patients were divided into two groups based on TOMM40 rs157581-G status, and clinical and imaging data were compared between the groups. TOMM40 rs157581-G carriers of AD showed decreased regional homogeneity in the left precuneus and decreased amplitude of low-frequency fluctuations in the bilateral temporal poles compared with noncarriers of AD. TOMM40 rs157581-G carriers of AD also showed increased functional connectivity between the right middle occipital gyrus and the left supramarginal gyrus and decreased connectivity between the left superior occipital gyrus and the right transverse temporal gyrus in comparison with TOMM40 rs157581-G noncarriers. We analyzed rs-fMRI characteristics of TOMM40 rs157581-G carriers of AD for the first time, which suggest that TOMM40 rs157581-G plays a harmful role in AD patients.

Structure of the mitochondrial import gate reveals distinct preprotein paths.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1-4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5-9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits, providing insight into critical features that areconserved in all eukaryotes1-3. Each Tom40 β-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.

Beyond ER: Regulating TOM-Complex-Mediated Import by Ubx2

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 (Mårtensson 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.

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

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

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

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

Recruitment of Cytosolic J-Proteins by TOM Receptors Promotes Mitochondrial Protein Biogenesis

SummaryMitochondria 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.

Epigenetic upregulation of miR-126 induced by heat stress contributes to apoptosis of rat cardiomyocytes by promoting Tomm40 transcription

TOMM40 is the channel-forming subunit of a translocase of the mitochondrial outer membrane (TOM) that is essential for protein transport into mitochondria. TOMM40 plays an important role in maintaining normal mitochondrial function. The correlation between occupational thermal exposure and mitochondria dysfunction has been demonstrated; however, nothing is known about the alteration and role of TOMM40 in response to environmental heat stress. In the present study, we showed that environmental thermal exposure upregulated microRNA miR-126, consequently reducing AU-rich element RNA-binding protein 1 (AUF1)-mediated SP1 mRNA degradation and increasing TOMM40 transcription, which in turn decreased the mitochondria membrane potential and apoptosis of cardiomyocytes. Mechanistically, miR-126 upregulation was attributed to heat stress-induced promoter demethylation via elevated TET2 (Tet methylcytosine dioxygenase 2) expression, while SP1 mRNA degradation was caused by decreased translation of AUF1 induced by miR-126. Moreover, TOMM40 transcription was upregulated via increasing its transcription factor SP1 resulting from AUF1 inhibition in the heat stress responses. The results of the present study increased our understanding of the role of miR-126 and TOMM40 in heat stressed cardiomyocytes.

Mitochondrial protein import - Functional analysis of the highly diverged Tom22 orthologue of Trypanosoma brucei

The β-barrel protein Tom40 and the α-helically anchored membrane protein Tom22 are the only universally conserved subunits of the protein translocase of the mitochondrial outer membrane (TOM). Tom22 has an N-terminal cytosolic and a C-terminal intermembrane space domain. It occurs in two variants: one typified by the yeast protein which has a cytosolic domain containing a cluster of acidic residues, and a shorter variant typified by the plant protein that lacks this domain. Yeast-type Tom22 functions as a secondary protein import receptor and is also required for the stability of the TOM complex. Much less is known about the more widespread short variant of Tom22, which is also found in the parasitic protozoan Trypanosoma brucei. Here we show that the intermembrane space domain of trypanosomal Tom22 binds mitochondrial precursor proteins and that it is essential for normal growth and mitochondrial protein import. Moreover, complementation experiments indicate that the intermembrane space domain cannot be replaced by the corresponding regions of the yeast or plant Tom22 orthologues. Lack or replacement of the short cytosolic domain, however, does not interfere with protein function. Finally, we show that only the membrane-spanning domain of trypanosomal Tom22 is essential for assembly of the trypanosomal TOM complex analogue.

The Import of Proteins into the Mitochondrion of Toxoplasma gondii

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

Dynamic organization of the mitochondrial protein import machinery.

Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

Blood glucose levels and cortical thinning in cognitively normal, middle-aged adults

Type II diabetes mellitus (DM) increases risk for cognitive decline and is associated with brain atrophy in older demented and non-demented individuals. We investigated (1) the cross-sectional association between fasting blood glucose level and cortical thickness in a sample of largely middle-aged, cognitively normal adults, and (2) whether these associations were modified by genes associated with both lipid processing and dementia. To explore possible modifications by genetic status, we investigated the interaction between blood glucose levels and the apolipoprotein E (APOE) ε4 allele and the translocase of the outer mitochondrial membrane (TOMM) 40 '523 genotype on cortical thickness. Cortical thickness measures were based on mean thickness in a subset of a priori-selected brain regions hypothesized to be vulnerable to atrophy in Alzheimer's disease (AD) (i.e., ‘AD vulnerable regions’). Participants included 233 cognitively normal subjects in the BIOCARD study who had a measure of fasting blood glucose and cortical thickness measures, quantified by magnetic resonance imaging (MRI) scans. After adjustment for age, sex, race, education, depression, and medical conditions, higher blood glucose was associated with thinner parahippocampal gyri (B=−0.002; 95% CI −0.004, −0.0004) and temporal pole (B=−0.002; 95% CI −0.004, −0.0001), as well as reduced average thickness over AD vulnerable regions (B=−0.001; 95% CI −0.002, −0.0001). There was no evidence for greater cortical thinning in ε4 carriers of the APOE gene or in APOE ε3/3 individuals carrying the TOMM40 VL/VL genotypes. When individuals with glucose levels in the diabetic range (≥126mg/dL), were excluded from the analysis, the associations between glucose levels and cortical thickness were no longer significant. These findings suggest that glucose levels in the diabetic range are associated with reduced cortical thickness in AD vulnerable regions as early as middle age.