Biology subjects

Gahura, O.

Publications and source records attributed to Gahura, O..

11 recordsLinked to original sources

bioRxiv2026

A pan-eukaryotic analysis of mitochondrial ribosomes uncovers major evolutionary transitions

Mitochondrial ribosomes (mitoribosomes) have diverged extensively both from their bacterial ancestors and across eukaryotes, and while many general trends have been identified, fundamental features of their evolution remain unclear. We analyzed sequences from 85 eukaryotes to chart protein composition across the evolution of mitochondrial translation. We both reconstruct the LECA mitoribosomal proteome as containing nearly the full bacterial protein complement and at least 23 proteins recruited after -proteobacterial endosymbiosis and describe the subsequent extensive but lineage-specific remodeling post LECA. We further uncover previously unrecognized homologies among several mitoribosomal proteins and unify and update the mitoribosomal nomenclature. Our data also support protein retargeting between organellar ribosomes, documenting remarkable intercompartmental structural compatibility. Together, these findings highlight the exceptional evolutionary plasticity of mitoribosomes and provide a comprehensive reference framework for future analysis.

evolutionary biology↗

bioRxiv2026

Alternative oxidase in trypanosomatids

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

biochemistry↗

bioRxiv2026

Interconnections with and within the trypanosomal respiratory chain revealed by complexome profiling

Proteins are frequently integrated into multicomponent complexes that execute the elaborate processes supporting life. Thus, a protein's function can only be defined by the company it keeps within a complex. Proteomes provide informative protein inventories but lack information about protein quaternary structures. Complexome profiling (CP) has been transformative in capturing the comprehensive population structure of complexes at a given moment within a cell. We have employed CP to chart the multiprotein complex landscape of two life cycle stages of Trypanosoma brucei. These data have allowed the observation of previously hidden interactions with and within the mitochondrial respiratory chain. We have found (1) an exceptional case of a SLC25 solute transporter that interacts with NADH dehydrogenase, (2) two ATP synthase subunit g paralogs that are intriguingly excluded from the enzyme's dimers, and (3) refined the known composition of ubiquinol:cytochrome c oxidoreductase by addition of missing subunits and removing an incorrectly assigned subunit, which more likely acts to insert the iron-sulfur co-factor into the complex. Further investigation into ubiquinol:cytochrome c oxidoreductase assembly revealed crosstalk between incorporation of its nuclear subunits with mitochondrial translation, possibly facilitating a hitherto unknown quality control mechanism. These discoveries demonstrate the power of our CP data for generation and testing of hypotheses about the mitochondrial and other organellar multiprotein complexes of T. brucei, a protist of medical and evolutionary importance.

cell biology↗

bioRxiv2026

A p32 family RNA editing factor acts in mitochondrial ribosome biogenesis

Biogenesis of mitochondrial ribosomes (mitoribosomes) in the unicellular parasite Trypanosoma brucei requires an exceptionally large toolkit of assembly factors, identified in stable precursors of large and small mitoribosomal subunits (mtLSU and mtSSU) by cryoEM. Here, using genetic modifications and proteomic characterization of the immunoprecipitated assemblosome, the earliest characterized mtSSU precursor, we determined that a cap of its distinctive protrusion of hitherto unknown composition consists of a p22 homotrimer. This protein was previously implicated in the uridine-insertion editing of the cytochrome c oxidase subunit II transcript. Our functional analysis confirmed this role but revealed that its ablation also causes a loss of mtSSU and a systemic reduction in mitochondrial translation, phenocopying the depletion of established mitoribosomal assembly factors. Consequently, the oxidative phosphorylation system and mitochondrial function are compromised. The p22 protein belongs to the p32 family. We showed that five of its six trypanosomal members are involved in mtSSU biogenesis. Notably, p32 proteins are associated with mitoribosomes in two other distant eukaryotic lineages. A eukaryote-wide mapping of p32 proteins documented that their presence correlates with the retention of mitochondrial genomes. Together, our findings redefine trypanosomal p22 as a dual-function coordinator of mitochondrial gene expression and reveal that the ancestral role of the p32 family is associated with mitochondrial translation.

molecular biology↗

bioRxiv2026

Distinct roles of three trypanosomal Oxa1 insertases in biogenesis of mitochondrial membrane complexes

The insertase Oxa1 is required for protein insertion into the inner mitochondrial membrane and for the biogenesis of oxidative phosphorylation complexes. While most eukaryotes encode one or two Oxa1 proteins, we identified three paralogs in Trypanosoma brucei: TbOxa1-1, TbOxa1-2, and TbOxa1-3. Knock-out of individual paralogs followed by phenotypic analyses and proteomic characterization of submitochondrial fractions revealed distinct functions. Respiratory chain complexes I and IV are primarily affected by loss of TbOxa1-1, whereas complex III and ATP synthase depend on TbOxa1-2; the ablation of TbOxa1-3 results in minor phenotypes in culture. In TbOxa1-2-depleted cells, ATP synthase biogenesis is compromised by the defective import or processing of the nuclear-encoded subunit-c, which also requires a rhomboid peptidase-like protein. Further, the ablation of TbOxa1-2 triggers accumulation of membrane proteins in the matrix, supporting its role in conservative sorting. Together, our results demonstrate that the trypanosomal Oxa1 machinery evolved a paralog-specific division of labor to manage a highly divergent mitochondrial membrane proteome.

biochemistry↗

bioRxiv2025

Mettl15-Mettl17 modulates the transition from early to late pre-mitoribosome

The assembly of the mitoribosomal small subunit involves folding and modification of rRNA, and its association with mitoribosomal proteins. This process is assisted by a dynamic network of assembly factors. Conserved methyltransferases Mettl15 and Mettl17 act on the solvent-exposed surface of rRNA. Binding of Mettl17 is associated with the early assembly stage, whereas Mettl15 is involved in the late stage, but the mechanism of transition between the two was unclear. Here, we integrate structural data from Trypanosoma brucei with mammalian homologs and molecular dynamics simulations. We reveal how the interplay of Mettl15 and Mettl17 in intermediate steps links the distinct stages of small subunit assembly. The analysis suggests a model wherein Mettl17 acts as a platform for Mettl15 recruitment. Subsequent release of Mettl17 allows a conformational change of Mettl15 for substrate recognition. Upon methylation, Mettl15 adopts a loosely bound state which ultimately leads to its replacement by initiation factors, concluding the assembly. Together, our results indicate that assembly factors Mettl15 and Mettl17 cooperate to regulate the biogenesis process, and present a structural data resource for understanding molecular adaptations of assembly factors in mitoribosome.

molecular biology↗

bioRxiv2023

Physiological variability in mitochondrial rRNA predisposes to metabolic syndrome

Metabolic syndrome is a growing concern in developed societies, and due to its polygenic nature, the genetic component is only slowly being elucidated. Common mitochondrial DNA sequence variants have been associated with symptoms of metabolic syndrome and may be relevant players in the genetics of metabolic syndrome. We investigate the effect of mitochondrial sequence variation on the metabolic phenotype in conplastic rat strains with identical nuclear but unique mitochondrial genomes, challenged by high-fat diet. We find that the variation in mitochondrial rRNA sequence represents a risk factor in insulin resistance development, which is caused by diacylglycerols accumulation induced by tissue-specific reduction of the oxidative capacity. These metabolic perturbations stem from the 12S rRNA sequence variation affecting mitochondrial ribosome assembly and translation. Our work demonstrates that physiological variation in mitochondrial rRNA might represent a relevant underlying factor in the progression of metabolic syndrome. Competing interestsThe authors declare that no competing interests exist.

physiology↗

bioRxiv2023

The ancestral shape of the access proton path of mitochondrial ATP synthases revealed by a split subunit-a

The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half-channel of mitochondrial ATP synthases are largely defined by a short N-terminal -helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the -helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This -helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The -helix blocks one of two proton routes found in Escherichia coli, resulting in the single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half-channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

biochemistry↗

bioRxiv2021

An ancestral interaction module promotes oligomerisation in divergent mitochondrial ATP synthases

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

molecular biology↗

bioRxiv2021

MICOS and F1FO-ATP synthase crosstalk is a fundamental property of mitochondrial cristae

Mitochondrial cristae are polymorphic invaginations of the inner membrane that are the fabric of cellular respiration. Both the Mitochondrial Contact Site and Cristae Organization System (MICOS) and the FOF1-ATP synthase are vital for sculpting cristae by opposing membrane bending forces. While MICOS promotes negative curvature at cristae junctions, dimeric FOF1- ATP synthase is crucial for positive curvature at cristae rims. Crosstalk between these two complexes has been observed in bakers yeast, the model organism of the Opisthokonta supergroup. Here, we report that this property is conserved in Trypanosoma brucei, a member of the Discoba supergroup that separated from Opisthokonta [~]2 billion years ago. Specifically, one of the paralogs of the core MICOS subunit Mic10 interacts with dimeric F1FO-ATP synthase, whereas the other core Mic60 subunit has a counteractive effect on F1FO- ATP synthase oligomerization. This is evocative of the nature of MICOS-F1FO-ATP synthase crosstalk in yeast, which is remarkable given the diversification these two complexes have undergone during almost 2 eons of independent evolution. Furthermore, we identified a highly diverged trypanosome homolog of subunit e, which is essential for the stability of F1FO-ATP synthase dimers in yeast. Just like subunit e, it is preferentially associated with dimers, interacts with Mic10 and its silencing results in severe defects to cristae and disintegration of F1FO-ATP synthase dimers. Our findings indicate that crosstalk between MICOS and dimeric F1FO-ATP synthase is a fundamental property impacting cristae shape throughout eukaryotes. ImportanceMitochondria have undergone profound diversification in separate lineages that have radiated since the last common ancestor of eukaryotes some eons ago. Most eukaryotes are unicellular protists, including etiological agents of infectious diseases like Trypanosoma brucei. Thus, the study of a broad range of protists can reveal fundamental features shared by all eukaryotes and lineage-specific innovations. Here we report that two different protein complexes, MICOS and F1FO-ATP synthase, known to affect mitochondrial architecture, undergo crosstalk in T. brucei, just as in bakers yeast. This is remarkable considering that these complexes have otherwise undergone many changes during their almost two billion years of independent evolution. Thus, this crosstalk is a fundamental property needed to maintain proper mitochondrial structure even if the constituent players considerably diverged.

cell biology↗

bioRxiv2020

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit in trypanosomes

Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes with reduced RNA and increased protein mass from Trypanosoma brucei, to provide insights into the biogenesis of mitoribosomal large subunit. Structural characterisation of a stable assembly intermediate revealed 22 assembly factors, some of which are also encoded in mammalian genomes. The assembly factors form a protein network that spans over 180 [A], shielding the ribosomal RNA surface. The entire central protuberance and L7/L12 stalk are not assembled, and require removal of the factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly, while the exit tunnel is blocked. Together, the extensive network of the factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

evolutionary biology↗