Mitochondria contain their own DNA (mtDNA) and a dedicated gene expression machinery. As the mitochondrial dimensions are close to the diffraction limit of classical light microscopy, the spatial distribution of mitochondrial proteins and in particular of mitochondrial mRNAs remains underexplored. Here, we establish single-molecule fluorescence in situ hybridization (smFISH) combined with STED and MINFLUX super-resolution microscopy (nanoscopy) to visualize individual mitochondrial mRNA molecules and associated proteins. STED nanoscopy reveals the spatial relationships between distinct mRNA species and proteins such as the RNA granule marker GRSF1, demonstrating adaptive changes in mRNA distribution and quantity in challenged mammalian cells and patient-derived cell lines. Notably, STED-smFISH shows the release of mRNAs during apoptosis, while MINFLUX reveals the folding of the mRNAs into variable shapes, as well as their spatial proximity to mitochondrial ribosomes. These protocols are transferable to various cell types and open new avenues for understanding mitochondrial gene regulation in health and disease.

The correct sorting of nascent ribosomal proteins from the cytoplasm to the nucleus or to mitochondria for ribosome production poses a logistical challenge for cellular targeting pathways. Here we report the discovery of a conserved mitochondrial avoidance segment (MAS) within the cytosolic ribosomal protein uS5 that resolves an evolutionary lethal conflict between the nuclear and mitochondrial targeting machinery. MAS removal mistargets uS5 to the mitochondrial matrix and disrupts the assembly of the cytosolic ribosome. The resulting lethality can be rescued by impairing mitochondrial import. We show that MAS triages nuclear targeting by disabling a cryptic mitochondrial targeting activity within uS5 and thereby prevents fatal capture by mitochondria. Our findings identify MAS as an essential acquisition by the primordial eukaryote that reinforced organelle targeting fidelity while developing an endosymbiotic relationship with its mitochondrial progenitor. Oborská-Oplová et al. report a conserved mitochondrial avoidance segment in the cytosolic ribosomal protein uS5 that prevents mistargeting of uS5 to the mitochondrial matrix and ensures cytosolic ribosomal assembly.

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.

T-cell receptor (TCR)-induced Ca2+ signals are essential for T-cell activation and function. In this context, mitochondria play an important role and take up Ca2+ to support elevated bioenergetic demands. However, the functional relevance of the mitochondrial-Ca2+-uniporter (MCU) complex in T-cells was not fully understood. Here, we demonstrate that TCR activation causes rapid mitochondrial Ca2+ (mCa2+) uptake in primary naive and effector human CD4+ T-cells. Compared to naive T-cells, effector T-cells display elevated mCa2+ and increased bioenergetic and metabolic output. Transcriptome and proteome analyses reveal molecular determinants involved in the TCR-induced functional reprogramming and identify signalling pathways and cellular functions regulated by MCU. Knockdown of MCUa (MCUaKD), diminishes mCa2+ uptake, mitochondrial respiration and ATP production, as well as T-cell migration and cytokine secretion. Moreover, MCUaKD in rat CD4+ T-cells suppresses autoimmune responses in an experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model. In summary, we demonstrate that mCa2+ uptake through MCU is essential for proper T-cell function and has a crucial role in autoimmunity. T-cell specific MCU inhibition is thus a potential tool for targeting autoimmune disorders.

Cristae are invaginations of the mitochondrial inner membrane that are crucial for cellular energy metabolism. The formation of cristae requires the presence of a protein complex known as MICOS, which is conserved across eukaryotic species. One of the subunits of this complex, MIC10, is a transmembrane protein that supports cristae formation by oligomerization. In Drosophila melanogaster, three MIC10-like proteins with different tissue-specific expression patterns exist. We demonstrate that CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Its loss destabilizes MICOS, disturbs cristae architecture, and reduces the life span and fertility of flies. We show that DmMIC10b has a unique ability to polymerize into bundles of filaments, which can remodel mitochondrial crista membranes. The formation of these filaments relies on conserved glycine and cysteine residues, and can be suppressed by the co-expression of other Drosophila MICOS proteins. These findings provide new insights into the regulation of MICOS in flies, and suggest potential mechanisms for the maintenance of mitochondrial ultrastructure.

Cellular proteostasis requires transport of polypeptides across membranes. Although defective transport processes trigger cytosolic rescue and quality control mechanisms that clear translocases and membranes from unproductive cargo, proteins that are synthesized within mitochondria are not accessible to these mechanisms. Mitochondrial-encoded proteins are inserted cotranslationally into the inner membrane by the conserved insertase OXA1L. Here, we identify TMEM126A as a OXA1L-interacting protein. TMEM126A associates with mitochondrial ribosomes and translation products. Loss of TMEM126A leads to the destabilization of mitochondrial translation products, triggering an inner membrane quality control process, in which newly synthesized proteins are degraded by the mitochondrial iAAA protease. Our data reveal that TMEM126A cooperates with OXA1L in protein insertion into the membrane. Upon loss of TMEM126A, the cargo-blocked OXA1L insertase complexes undergo proteolytic clearance by the iAAA protease machinery together with its cargo.

Mitochondria are the powerhouses of the cell as they produce the majority of ATP with their oxidative phosphorylation (OXPHOS) machinery. The OXPHOS system is composed of the F1 Fo ATP synthase and four mitochondrial respiratory chain complexes, the terminal enzyme of which is the cytochrome c oxidase (complex IV) that transfers electrons to oxygen, generating water. Complex IV comprises of 14 structural subunits of dual genetic origin: while the three core subunits are mitochondrial encoded, the remaining constituents are encoded by the nuclear genome. Hence, the assembly of complex IV requires the coordination of two spatially separated gene expression machinery. Recent efforts elucidated an increasing number of proteins involved in mitochondrial gene expression, which are linked to complex IV assembly. Additionally, several COX1 biogenesis factors have been intensively biochemically investigated and an increasing number of structural snapshots shed light on the organization of macromolecular complexes such as the mitoribosome or the cytochrome c oxidase. Here, we focus on COX1 translation regulation and highlight the advanced understanding of early steps during COX1 assembly and its link to mitochondrial translation regulation.

Mitochondria are essential eukaryotic double-membrane organelles. The convoluted mitochondrial inner membrane forms highly organized invaginations, termed cristae, which are crucial for energy metabolism. Cristae formation requires MICOS, a conserved hetero-oligomeric inner membrane complex. The MICOS core subunit MIC10 is a small transmembrane protein that oligomerizes through highly conserved glycine-rich motifs to control cristae formation. Sequence alignments show that D. melanogaster exhibits three MIC10-like proteins with different tissue-specific expression patterns. Here, we show that the ubiquitously expressed Dmel_CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Loss of DmMIC10b disturbs cristae architecture of mitochondria and reduces the life-span and fertility of flies. Moreover, using fluorescence nanoscopy and electron tomography, we demonstrate that despite its high similarity to the MIC10 proteins from yeast and humans, DmMIC10b exhibits the unique ability to polymerize into elongated filaments upon overexpression. DmMIC10b filaments form bundles which accumulate in the intermembrane space and alter the shape of mitochondrial cristae membranes. We show that the formation of the filaments relies on conserved glycine and cysteine residues and is suppressed by co-expression of other MICOS proteins. Thereby, our findings provide new insights into the regulation of MICOS in flies.### Competing Interest StatementThe authors have declared no competing interest.

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria.

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1(-/-) cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.

Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis. The performance of clinicopathologic parameters and molecules as prognostic factors remains limited and inconsistent. The present study aimed to construct a multi-molecule biomarker panel to more accurately predict post-resectional prognosis of PDAC patients.Firstly, a novel computational strategy integrating prognostic evidence from omics and literature on the basis of bioinformatics prediction (CIPHER) to generate the network, was designed to systematically identify potential high-confidence PDAC-related prognostic candidates. After specimens from 605 resected PDAC patients were retrospectively collected, 23 candidates were detected immunohistochemically in tissue-microarrays for the development cohort to construct a multi-molecule panel. Lastly, the panel was validated in two independent cohorts.According to the constructed five-molecule panel, disease-specific survival (DSS) was significantly poorer in high-risk patients than in low-risk ones in development cohort (HR 2.15, 95%CI 1.51–3.05, P<0.0001; AUC 0.67). In two validation cohorts, similar significant differences between the two groups were also observed (HR 3.18 and 3.31, 95%CI 1.89–5.37 and 1.78–6.16, All P<0.0001; AUC 0.72 and 0.73). In multivariate analyses, this panel was the sole prognosticator that was significant in each cohort. Furthermore, its predictive power for long-term survival, higher than its individual constituents, could be largely enhanced by combination with traditional clinicopathological variables. Finally, adjuvant chemotherapy (ACT) correlated with better DSS only in high-risk patients, uni- and multi-variately, in all the cohorts.The novel prognostic panel developed by a systematically network-based strategy presents strong ability in prediction of post-resectional survival of PDAC patients. Furthermore, panel-defined high-risk patients might benefit more from ACT.

Chronic pain is a prevalent medical problem, and its molecular basis remains poorly understood. Here, we demonstrate the significance of the transmembrane protein (Tmem) 160 for nerve injury-induced neuropathic pain. An extensive behavioral assessment suggests a pain modality- and entity-specific phenotype in male Tmem160 global knockout (KO) mice: delayed establishment of tactile hypersensitivity and alterations in self-grooming after nerve injury. In contrast, Tmem160 seems to be dispensable for other nerve injury-induced pain modalities, such as non-evoked and movement-evoked pain, and for other pain entities. Mechanistically, we show that global KO males exhibit dampened neuroimmune signaling and diminished TRPA1-mediated activity in cultured dorsal root ganglia. Neither these changes nor altered pain-related behaviors are observed in global KO female and male peripheral sensory neuron-specific KO mice. Our findings reveal Tmem160 as a sexually dimorphic factor contributing to the establishment, but not maintenance, of discrete nerve injury-induced pain behaviors in male mice.

Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.

The mitochondrial proteome is built and maintained mainly by import of nuclear‐encoded precursor proteins. Most of these precursors use N‐terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2–SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis.

The mitochondrial presequence translocation machinery (TIM23 complex) is conserved between the yeast Saccharomyces cerevisiae and humans; however, functional characterization has been mainly performed in yeast. Here, we define the constituents of the human TIM23 complex using mass spectrometry and identified ROMO1 as a new translocase constituent with an exceptionally short half-life. Analyses of a ROMO1 knockout cell line revealed aberrant inner membrane structure and altered processing of the GTPase OPA1. We show that in the absence of ROMO1, mitochondria lose the inner membrane YME1L protease, which participates in OPA1 processing and ROMO1 turnover. While ROMO1 is dispensable for general protein import along the presequence pathway, we show that it participates in the dynamics of TIM21 during respiratory chain biogenesis and is specifically required for import of YME1L. This selective import defect can be linked to charge distribution in the unusually long targeting sequence of YME1L. Our analyses establish an unexpected link between mitochondrial protein import and inner membrane protein quality control.

The mitochondrial genome encodes for thirteen core subunits of the oxidative phosphorylation system. These proteins assemble with imported proteins in a modular manner into stoichiometric enzyme complexes. Assembly factors assist in these biogenesis processes by providing co-factors or stabilizing transient assembly stages. However, how expression of the mitochondrial-encoded subunits is regulated to match the availability of nuclear-encoded subunits is still unresolved. Here, we address the function of MITRAC15/COA1, a protein that participates in complex I biogenesis and complex IV biogenesis. Our analyses of a MITRAC15 knockout mutant reveal that MITRAC15 is required for translation of the mitochondrial-encoded complex I subunit ND2. We find that MITRAC15 is a constituent of a ribosome-nascent chain complex during ND2 translation. Chemical crosslinking analyses demonstrate that binding of the ND2-specific assembly factor ACAD9 to the ND2 polypeptide occurs at the C-terminus and thus downstream of MITRAC15. Our analyses demonstrate that expression of the founder subunit ND2 of complex I undergoes regulation. Moreover, a ribosome-nascent chain complex with MITRAC15 is at the heart of this process.