Mitochondrial сomplexome of etiolated pea shoots

Studies into mitochondrial сomplexomes in various organisms provide an insight into the native organization of proteins and metabolic pathways in the organelles of the subject under study. “Complexome” is a relatively recent concept describing the proteome of protein complexes, supercomplexes, and oligomeric proteins. Complexome analysis is performed using current electrophoretic and mass spectrometric techniques, in particular, by two-dimensional electrophoresis (2D BN/SDS-PAGE) in combination with mass spectrometry (MS). Unlike 2D IEF/SDS-PAGE, this method enables analysis of not only hydrophilic proteins of the mitochondrial matrix, but also membrane proteins and their associations, thus expanding the possibilities of studying the organelle proteome. In the present work, the complexome of etiolated pea shoots was studied for the first time using 2D BN/SDS-PAGE followed by MALDI-TOF MS. To this end, 145 protein spots excised from the gel were analyzed; 110 polypeptides were identified and assigned to different functional groups. A densitometric analysis revealed that the major protein group comprised the enzymes of the mitochondrial energy system (1), accounting for an average of 43% of the total polypeptide content. The remaining 57% was primarily distributed among the following functional categories: pyruvate dehydrogenase complex and citric acid cycle (2); amino acid metabolism (3); nucleic acid processing (4); protein folding (5); antioxidant protection (6); carrier proteins (7); other proteins (8); proteins having unknown functions (9). The obtained data indicate the complex organization of the pea proteome. In addition to the enzymes of the OXPHOS system, the proteins of other functional categories are found to form supramolecular structures. It is suggested that the presence of proteins from other cellular compartments may indicate the interaction of mitochondria with the enzymes or structures of corresponding organelles. In general, the obtained data on the pea complexome represent a kind of a mitochondrial “passport” that reflects the native state of the proteome of organelles corresponding to their physiological status.

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. 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.

Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators

Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrial-derived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner.

The subcompartmented oxphosomic model of the phosphorylating system organization in mitochondria

The oxidative phosphorylation (OXPHOS) system of mitochondria supports all the vitally important energyconsuming processes in eukaryotic cells, providing them with energy in the form of ATP. OXPHOS enzymes (complexes I–V) are located in the inner mitochondrial membrane, mainly in the cristae subcompartment. At present, there is a large body of data evidencing that the respiratory complexes I, III2 and IV under in vivo conditions can physically interact with each other in diverse stoichiometry, thereby forming supercomplexes. Despite active accumulation of knowledge about the structure of the main supercomplexes of the OXPHOS system, its physical and functional organization in vivo remains unclear. Contemporary models of the OXPHOS system’s organization in the inner membrane of mitochondria are contradictory and presume the existence of either highly organized respiratory strings, or, by contrast, a set of randomly dispersed respiratory supercomplexes and complexes. Furthermore, it is assumed that ATP-synthase (complex V) does not form associations with respiratory enzymes and operates autonomously. Our latest data obtained on mitochondria of etiolated shoots of pea evidence the possibility of physical association between the respiratory supercomplexes and dimeric ATP-synthase. These data have allowed us to reconsider the contemporary concept of the phosphorylation system organization and propose a new subcompartmented oxphosomic model. According to this model, a substantial number of the OXPHOS complexes form oxphosomes, which in a definite stoichiometry include complexes I–V and are located predominantly in the cristae subcompartment of mitochondria in the form of highly organized strings or patches. These suprastructures represent “mini-factories” for ATP production. It is assumed that such an organization (1) contributes to increasing the efficiency of the OXPHOS system operation, (2) involves new levels of activity regulation, and (3) may determine the inner membrane morphology to some extent. The review discusses the proposed model in detail. For a better understanding of the matter, the history of development of concepts concerning the OXPHOS organization with the emphasis on recent contemporary models is briefly considered. The principal experimental data accumulated over the past 40 years, which confirm the validity of the oxphosomic hypothesis, are also provided.

MRX8, the conserved mitochondrial YihA GTPase family member is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae

The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multi-subunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress is not sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8 is a bonafide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggest a universal role of MRX8 family of proteins in regulating mitochondrial function.

Tackling Dysfunction of Mitochondrial Bioenergetics in the Brain

Oxidative phosphorylation (OxPhos) is the basic function of mitochondria, although the landscape of mitochondrial functions is continuously growing to include more aspects of cellular homeostasis. Thanks to the application of -omics technologies to the study of the OxPhos system, novel features emerge from the cataloging of novel proteins as mitochondrial thus adding details to the mitochondrial proteome and defining novel metabolic cellular interrelations, especially in the human brain. We focussed on the diversity of bioenergetics demand and different aspects of mitochondrial structure, functions, and dysfunction in the brain. Definition such as ‘mitoexome’, ‘mitoproteome’ and ‘mitointeractome’ have entered the field of ‘mitochondrial medicine’. In this context, we reviewed several genetic defects that hamper the last step of aerobic metabolism, mostly involving the nervous tissue as one of the most prominent energy-dependent tissues and, as consequence, as a primary target of mitochondrial dysfunction. The dual genetic origin of the OxPhos complexes is one of the reasons for the complexity of the genotype-phenotype correlation when facing human diseases associated with mitochondrial defects. Such complexity clinically manifests with extremely heterogeneous symptoms, ranging from organ-specific to multisystemic dysfunction with different clinical courses. Finally, we briefly discuss the future directions of the multi-omics study of human brain disorders.

Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency

Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the patient’s cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were quantified by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a compensatory mechanism in COX4-1 deficiency.

A Mitochondrial Polymorphism Alters Immune Cell Metabolism and Protects Mice from Skin Inflammation

Several genetic variants in the mitochondrial genome (mtDNA), including ancient polymorphisms, are associated with chronic inflammatory conditions, but investigating the functional consequences of such mtDNA polymorphisms in humans is challenging due to the influence of many other polymorphisms in both mtDNA and the nuclear genome (nDNA). Here, using the conplastic mouse strain B6-mtFVB, we show that in mice, a maternally inherited natural mutation (m.7778G > T) in the mitochondrially encoded gene ATP synthase 8 (mt-Atp8) of complex V impacts on the cellular metabolic profile and effector functions of CD4+ T cells and induces mild changes in oxidative phosphorylation (OXPHOS) complex activities. These changes culminated in significantly lower disease susceptibility in two models of inflammatory skin disease. Our findings provide experimental evidence that a natural variation in mtDNA influences chronic inflammatory conditions through alterations in cellular metabolism and the systemic metabolic profile without causing major dysfunction in the OXPHOS system.

Schizosaccharomyces pombe Ppr10 is required for mitochondrial translation

ABSTRACT The mitochondrial genome encodes key components of the oxidative phosphorylation (OXPHOS) system, whose expression is essential for mitochondrial functions. We have previously shown that deletion of the Schizosaccharomyces pombe ppr10 encoding a pentatricopeptide repeat protein severely reduces the mature levels of intron-containing mitochondrial transcripts cox1 and cob1, and severely impairs mitochondrial translation. In this study, we examined the possibility that the reduced levels of Cox1 and Cob1 proteins in cells were due to lowered levels of cox1 and cob1 mRNAs. We found that deletion of ppr10 did not affect the levels of mature cox1 and cob1 mRNAs in a mitochondrial intronless background. However, synthesis of Cox1 and Cob1 proteins were still severely affected by deletion of ppr10 in a mitochondrial intronless background. Consistent with this, we found that deletion of mitochondrial introns could not rescue the respiratory growth defect of Δppr10 cells. Our results reveal that Ppr10 is not required for the stability of cox1 and cob1 mRNAs, and provide further support for the idea that Ppr10 plays a critical role in mitochondrial translation.