Analysis of mitochondrial translation using click chemistry.

Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach.

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).

MIC10b can polymerize into cristae-shaping filaments.

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.

Identification of TMEM126A as OXA1L-interacting protein reveals cotranslational quality control in mitochondria.

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.

Cytochrome c oxidase biogenesis - from translation to early assembly of the core subunit COX1.

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 F F 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.

Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control.

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.

HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation.

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 HCLS1-associated 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.

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.

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.

Tmem160 contributes to the establishment of discrete nerve injury-induced pain behaviors in male mice.

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.

Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.

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).

An in vitro system to silence mitochondrial gene expression.

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.

Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery.

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.

Ribosome-Targeting Antibiotics Impair T Cell Effector Function and Ameliorate Autoimmunity by Blocking Mitochondrial Protein Synthesis.

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease.

Functional coupling of presequence processing and degradation in human mitochondria.

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.

COA6 Facilitates Cytochrome c Oxidase Biogenesis as Thiol-reductase for Copper Metallochaperones in Mitochondria.

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 Cu site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for Cu center formation.

MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome-nascent chain complex.

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.

Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7.

Nuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein cytochrome oxidase assembly factor 7 (COA7), or RESpiratory chain Assembly 1 (RESA1) factor that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency.

ROMO1 is a constituent of the human presequence translocase required for YME1L protease import.

The mitochondrial presequence translocation machinery (TIM23 complex) is conserved between the yeast 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.

The codon sequences predict protein lifetimes and other parameters of the protein life cycle in the mouse brain.

The homeostasis of the proteome depends on the tight regulation of the mRNA and protein abundances, of the translation rates, and of the protein lifetimes. Results from several studies on prokaryotes or eukaryotic cell cultures have suggested that protein homeostasis is connected to, and perhaps regulated by, the protein and the codon sequences. However, this has been little investigated for mammals in vivo.

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions.

The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses.

Sensing the Stress: A Role for the UPR and UPR in the Quality Control of Mitochondria.

Mitochondria exist as compartmentalized units, surrounded by a selectively permeable double membrane. Within is contained the mitochondrial genome and protein synthesis machinery, required for the synthesis of OXPHOS components and ultimately, ATP production. Despite their physical barrier, mitochondria are tightly integrated into the cellular environment.

COX16 promotes COX2 metallation and assembly during respiratory complex IV biogenesis.

Cytochrome oxidase of the mitochondrial oxidative phosphorylation system reduces molecular oxygen with redox equivalent-derived electrons. The conserved mitochondrial-encoded COX1- and COX2-subunits are the heme- and copper-center containing core subunits that catalyze water formation. COX1 and COX2 initially follow independent biogenesis pathways creating assembly modules with subunit-specific, chaperone-like assembly factors that assist in redox centers formation.

The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis.

The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the Cu-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network.

TORC1-mediated sensing of chaperone activity alters glucose metabolism and extends lifespan.

Protein quality control mechanisms, required for normal cellular functioning, encompass multiple functions related to protein production and maintenance. However, the existence of communication between proteostasis and metabolic networks and its underlying mechanisms remain elusive. Here, we report that enhanced chaperone activity and consequent improved proteostasis are sensed by TORC1 via the activity of Hsp82.