Open Biology

Heterozygous de novo missense mutations in the EEF1A2 gene encoding translation elongation factor eEF1A2 result in neurodevelopmental disorders, typically characterised by early onset epilepsy and intellectual disability (ID). The E122K mutation is the most commonly reported missense mutation and is amongst the more severe in terms of epilepsy and ID. Here we made use of a recently developed mouse model which recapitulates the E122K mutation to examine how mutations in EEF1A2 might disrupt neuronal gene expression. Primary neurons from mutant mice and transfected HEK293T cells were used to examine effects on global protein synthesis. In contrast to previous reports, we were unable to detect a change in global protein synthesis using either of two different assay systems. TRAP-seq and mass spectrometry were then employed to study the effects of the mutation on the translatome and proteome respectively. These analyses revealed perturbation of expression of a subset of genes, with a slight skew towards downregulation, particularly for longer transcripts. Further analysis indicated a down regulation of proteins involved in synaptic function in both the translatomic and proteomic datasets.

In trypanosmatids genes, transcribed by RNA polymerase II do not have canonical promoters and are organized into directional gene clusters that mature into monocistronic transcripts by a co-transcriptional process known as trans-splicing. Even though gene expression is regulated mainly post-transcriptionally, it is currently understood that chromatin and epigenetics are also involved in this regulation. In eukaryotes, specific signals are normally required for the occurrence of an appropriate transcription initiation. Among them, trimethylation of histone H3 in lysine 4 is the most conserved signal normally detected at transcription start sites of actively transcribed genes. Unlike many model organisms, trypanosomes do not have defined promoters. Instead, transcription initiates in a bidirectional manner from dispersed regions coincident with divergent strand switch regions located between directional gene clusters (DGCs). In T. cruzi, H3K4me3 was observed at the origins of transcription coincident with divergent strand switch regions (dSSRs) in epimastigotes, but it has not been mapped throughout the whole genome at base-pair resolution or in other life stages so far. Here, we set up the CUT&RUN technique for T. cruzi epimastigotes and trypomastigotes. Consistent with a predominant post-transcriptional regulation along the life cycle, we did not find significant differences between life stages. We corroborated that H3K4me3 is enriched at dSSR adjacent to actively expressed DGCs. Moreover, we noticed that this histone mark exhibits different patterns that correlate with the genomic span of the transcription initiation regions and with transcriptional activity. Furthermore, we unveiled that the most actively transcribed DGCs are associated with shorter dSSRs and are located within the core compartment of the genome displaying a more accessible chromatin.

Molecular motors travelling along DNA introduce positive supercoils that present as barriers to replication leading to genome instability. To counter these, bacterial cells express DNA gyrase, a topoisomerase that introduces negative supercoils. While much is known about DNA gyrase from genetic and in vitro biochemical studies, the spatiotemporal dynamics of this enzyme remain a mystery. Only recently have we been able to observe the in vivo spatiotemporal dynamics down to single molecule level using advanced super-resolution microscopy techniques. We used Slimfield microscopy, a cutting-edge molecule microscopy technique to address the gap in our knowledge. We analysed a dual fluorescently labelled Escherichia coli strain expressing the replisome marker DnaN-mCherry along with mYPet-GyrB as the enzyme marker. We performed sequential Slimfield microscopy of the labelled proteins from the same strain and analysed in vivo GyrB dynamics in live E. coli cells in relation to the replisome. We find that the majority of replisomes are associated with GyrB. Inhibition of gyrase activity reduces the proportion of replisomes associated with GyrB. Interestingly, GyrB behaviour is distinct from that observed for GyrA in a previous study. Our results reveal the previously unknown dynamics of GyrB inside living bacterial cells highlighting the advantages of in vivo single molecule investigations. Our findings also demonstrate the importance of analysing all subunits of a functional enzyme complex to gain comprehensive understanding of its in vivo mechanisms. This study demonstrates the utility of single-molecule super-resolved microscopy as a valuable underpinning technology to understand in vivo behaviour of biomedically important molecules. Our insights will help impact discovery and development of novel antibiotics that interfere with gyrase function, thus contributing to tackling the growing problem of antimicrobial resistance.

Polyploid cells, which contain more than two copies of the genome, are widely present across plants and animals, where they are often found in tissues with high biosynthetic and metabolic demands, such as the mammalian liver and placenta. While somatic polyploidy is frequently associated with increased cell growth and biosynthetic capacity, unscheduled polyploidization in cell types that are not normally programmed to become polyploid is often linked to reduced cellular fitness and genome instability. To understand whether these divergent outcomes stem from distinct immediate cellular responses to increased ploidy, we systematically compared the early consequences of polyploidization across naturally occurring and experimentally induced systems. Specifically, we examined physiological polyploid cells in the Caenorhabditis elegans intestine and human hepatocyte organoids, alongside unscheduled polyploid human retinal pigment epithelial (RPE1) cells generated through cytokinesis failure. Using quantitative imaging, flow cytometry, and FUCCI based cell-cycle reporters we measured cell size and protein translation dynamics during G1 in diploid and polyploid cells. Across all systems, we observed a strikingly conserved relationship between ploidy, cell size, and biosynthetic capacity: both cell size and protein translation showed similar scaling patterns after polyploidization, regardless of whether polyploidization occurred as part of normal development or by inducing cytokinesis failure. These findings indicate that the immediate cellular response to increased ploidy is broadly similar across contexts. However, in contrast to unscheduled polyploid RPE1 cells, polyploid human hepatocytes extend their G1 phase, leading to a higher accumulation of proteins before cell-cycle progression. Together, our findings suggest that polyploidization elicits similar growth responses across contexts, and that cell-type specific cell-cycle adaptations may determine whether polyploidy becomes advantageous or deleterious.

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (~130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

Microalgal metabolism relies on their chloroplasts, and involves both nucleus and plastidial-encoded proteins of various evolutionary origins. The plastidial ATP synthase complex is a key player in photosynthesis, and has been extensively studied in plants. However, our knowledge in other photosynthetic eukaryotes remains limited, despite their importance in marine environments. Here, we report the characterisation of a novel homologue of the F-type ATP synthase alpha subunit, hereby named xATPA, widespread in microalgae but absent from other photosynthetic organisms. Comparisons of xATPA sequences and predicted structures revealed a specific feature, the bump domain, and highlighted the absence of an ATP-binding site. We assessed xATPA prevalence in microalgae in the global ocean using environmental data from Tara Oceans, with a particular focus on diatoms, and demonstrate that its expression is associated with polar summer conditions. Using a reverse genetic approach in the model diatom Phaeodactylum tricornutum, we show that xATPAP t has a plastidial localisation, and that xATPA KO mutants exhibit growth deficiencies in a combination of low temperature, low salinity and constant light, consistent with environmental analysis. Surprisingly, both RNAseq and physiological assays suggest that xATPA is not involved in ATP synthase functions. On the other hand, xATPA interacts with other F1 ATP synthase subunits in vitro, which we suggest forms transient unassembled complexes. This study hence represents a comprehensive analysis of a novel protein from the environment to the lab, and reveals a new player in the plastidial physiology of eukaryotic microalgae.

Effective communication between cells is essential for the typical development and behaviour of an organism. In this context, gap junctions represent the most universally preserved components at cellular membranes of multicellular organisms, facilitating metabolic and electrical connections between cells. Disruptions in these junctions have been linked to various developmental abnormalities and pathological conditions in humans. The invertebrate gap junction proteins, referred to as innexins, exhibit conserved cellular and molecular mechanisms of functioning with their vertebrate counterparts, known as connexins. Consequently, they provide valuable means for studying and understanding the functions of gap junctions in development. In the Drosophila embryo, innexin-2 is expressed in the amnioserosa and ectoderm, where it is required for epithelial morphogenesis. Genetic depletion of innexin-2 results in cuticular defects and embryonic lethality. Pannier, a GATA family transcription factor, is a key regulator of dorsal tissue development in Drosophila and is expressed in the amnioserosa, dorsal ectoderm and the dorsal vessel during embryogenesis. Pannier mutants exhibit defects in dorsal closure, cuticle formation, and cardiac specification. Although substantial evidence from vertebrate systems indicate that connexin expression is regulated by transcription factors such as GATA4, Nkx2.5, Tbx2, Tbx3, and Tbx5, whether a similar regulatory relationship exists between these transcription factors and gap junction proteins in Drosophila remains unknown. In this study, we investigate how innexin mediated intercellular communication impacts pannier dependent morphogenetic processes during Drosophila embryogenesis.

Female reproduction is an energetically expensive process, so species evolve to balance survival with reproductive output. Many female organisms can temporarily pause their reproduction, including egg development, in response to physiological stress. The cellular mechanisms initiating and maintaining a stress-induced pause in oogenesis have been most extensively studied in Drosophila melanogaster. While the molecular control of paused oogenesis in response to starvation have been well characterized in flies, it remains unknown if these mechanisms are shared by other species with regulated pauses in oogenesis. Eusocial insects are characterized by a reproductive division of labor, with colonies of reproductive queens and sterile female workers. The social paper wasp, Polistes, has a dynamic dominance-based hierarchy for queen status. Worker Polistes are kept sterile by a combination of social and nutritional stressors. Here, we establish Polistes as a model to explore adult female reproductive plasticity. Through immunohistochemistry we have directly compared the Drosophila and Polistes ovarian structure and identified critical regions of the ovary in wasps that undergo regulated cell elimination during reproductive pause in flies. By comparing tissue structure, cell organization and rates of cell death between Polistes queens and workers we identified apoptosis as a key regulator maintaining worker sterility. Critically, this mechanism appears to be partially conserved with that in Drosophila. Finally, we find that changes in the timing and location of cell death in Polistes workers implicate oocyte identity and oocyte growth as additional potential regulators of temporary disruption of oogenesis.

In most organisms, all cells inherit the same genome, and many mechanisms exist to preserve its integrity across cell divisions. Programmed DNA elimination (PDE), the targeted removal of specific genomic regions from somatic cell lineages during early embryogenesis, is a striking exception. Since its discovery in parasitic nematodes over a century ago, PDE has been observed in diverse eukaryotes, including ciliates, arthropods, and vertebrates. However, the mechanisms, functions, and evolutionary origins of PDE remain poorly understood. Here, we describe the discovery of PDE in three species of the free-living nematode genus Caenorhabditis. Multiple genomic regions are precisely eliminated from somatic cells during early embryogenesis, resulting in chromosome fragmentation and the loss of key germline genes. The sites of elimination are strongly associated with conserved sequence motifs that likely direct DNA breakage. Comparative analyses indicate that PDE was present in the last common ancestor of Caenorhabditis and subsequently lost early during the evolution of many species, including C. elegans. The presence of PDE in the ancestors of one of biology's most important model organisms, together with recent discoveries in other eukaryotic lineages, reveals PDE to be a far more widespread and significant feature of evolution and development than previously recognised.

The MIKCc subfamily of MADS-box proteins play a key role in flowering plant reproduction, specifying and patterning the floral organs. MIKCC genes have been identified in non-flowering plants, the number increasing with the amount of whole-genome sequencing information available, but MIKCc functions outside flowering plants are less well understood. In this study, we have cloned and sequenced 14 of the 21 MIKCc genes in the model fern Ceratopteris richardii and identified expressed cDNAs/transcripts for a further 6 genes, extending and correcting previous genome-and transcriptome analysis. We have identified that the majority of Ceratopteris MIKCc genes are expressed in the reproductive sporophyte, with some genes showing reproductive specificity. Using protein alignments and structural modelling, we have shown that Ceratopteris MIKCC proteins are structurally diverse, with over half the proteins possessing extended regions N-terminal to the MADS DNA binding domain, suggesting divergent functions or regulation.

The heterotrimeric G-protein transducin (Gt) is among the key proteins mediating phototransduction in rod and cone cells of the vertebrate retina. Even though this protein has been extensively characterized in mammals, little is known about its expression patterns in migratory songbirds. Here we characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. The mechanism underlying magnetoreception is not fully understood, but one well-supported hypothesis involves a radical-pair formation in the blue light receptor cryptochrome type 4a. The - and {gamma}-subunits of cone specific transducin have been identified as possible interaction partners of cryptochrome 4a. Therefore, we analysed the expression patterns of various G-protein subunits in bird photoreceptors. Specifically, we combined single cell RNA sequencing and immunohistochemistry, and tested for protein interaction by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. We show that genes for G-protein subunits GNB1 and GNB3 (coding for Gt{beta}1 and Gt{beta}3, respectively) are predominantly expressed in rods and cones. Among {gamma}-subunits, GNGT2 (coding for Gt{gamma}T2) was the principal isoform in cones, whereas GNG11 (coding for Gt{gamma}11) was associated with rods. In contrast, we did not detect GNG10 (coding for Gt{gamma}10) expression in either photoreceptor type. Interaction assays demonstrated that all three {beta}{gamma} combinations; {beta}{gamma}T2, {beta}{gamma}10, and {beta}{gamma}11, can associate in vitro. These findings indicate that {beta}{gamma} dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. Furthermore, the absence of GNG10 expression in rods and cones does not support a role of this {gamma}-subunit in photoreceptor-based magnetoreception.

Mutations in chromosome alignment maintaining phosphoprotein 1 (CHAMP1) have been linked to neurodevelopmental disorders characterized by intellectual disability, developmental delay, and autism spectrum disorder. However, the cellular and electrophysiological mechanisms by which CHAMP1 mutations disrupt human neuronal development remain poorly understood. In the present study, we used patient-derived induced pluripotent stem cells (iPSCs) carrying two pathogenic CHAMP1 mutations and generated neural progenitor cells (NPCs) and excitatory neurons to investigate the effects of each mutation on neuronal maturation and function, DNA repair, and gene expression. Proliferative capacity and DNA repair dysfunction operate in a CHAMP1 dose-dependent manner. Whole-cell patch-clamp electrophysiology revealed that CHAMP1 mutant neurons exhibit significant alterations in intrinsic membrane properties during early developmental stages, including depolarized resting membrane potential, reduced action potential firing, and impaired waveform kinetics. These functional deficits were accompanied by reduced sodium and potassium current densities, suggesting impaired ion channel accumulation during neuronal maturation. Furthermore, recordings of spontaneous excitatory postsynaptic currents indicated altered synaptic activity and reduced proportions of synaptically active neurons. Morphological analyses revealed deficits in neurite outgrowth and branching, consistent with delayed neuronal maturation. Single-nucleus transcriptomic profiling further revealed delayed developmental trajectories and mutation-specific dysregulation of synaptic gene programs enriched for autism, ADHD, and epilepsy risk genes. Together, these findings demonstrate that CHAMP1 mutations disrupt multiple aspects of neuronal development, including homologous recombination (HR) dysfunction in NPCs, membrane excitability, ion channel function, and synaptic connectivity. Our results provide insights into the neurobiological consequences of CHAMP1 mutations and establish patient-derived neurons as a platform to investigate cellular pathophysiology and potential therapeutic strategies for CHAMP1-associated neurodevelopmental disorders.

Histone proteins, which reside in the nuclei of eukaryotic cells, are involved in diverse cellular processes. The core histone H4 serves as a structural component of the nucleosome. Patients carrying mutations in H4Clustered histone (H4C) genes exhibit a broad spectrum of developmental abnormalities, including short stature, microcephaly, intellectual disability, growth retardation, and digital anomalies. However, the impact of H4 mutations on mammalian embryogenesis remains largely unclear. Here, we demonstrate that histone H4C genes play crucial roles in skeletal development and cortical neurogenesis. We found that mRNAs of the histone H4C gene family are specifically expressed in proliferating progenitor cells in the developing mouse neocortex and in human induced pluripotent stem cell-derived cortical organoids. CRISPR-mediated disruption of H4C3 in mice caused severe defects in skeletal formation and neocortical neurogenesis. Furthermore, overexpression of a mutant form of H4C3 resulted in altered expression of genes associated with cellular migration and motility. Together, these findings suggest that histone H4 plays a critical role in regulating the balance between proliferation and differentiation during mammalian embryonic development, thereby explaining the broad spectrum of patient phenotypes.

Cyclin-dependent kinases (CDKs) control eukaryotic cell-cycle progression by phosphorylating specific substrates with substrate recognition often involving cyclin-specific docking interactions. However, in minimal cell cycle control systems driven by a single cyclin-CDK complex, how docking interactions contribute to the differential timing of substrate phosphorylation remains unclear. Here, we used AlphaFold-Multimer to systematically predict interactions between the fission yeast mitotic cyclin-CDK fusion Cdc13-L-Cdc2 and its known in vivo CDK substrates. We found that many substrates are predicted to interact with the cyclin hydrophobic patch, and have identified a previously uncharacterised docking motif, [FVIPWGLAM](x)xER[LMV] (ERL motif), with features consistent with an atypical RxL motif. We show that ERL motifs can functionally substitute for canonical RxL motifs to promote phosphorylation of a model CDK substrate by Cdc13-Cdc2, while the S-phase cyclin-CDK Cig2-Cdc2 was found to preferentially phosphorylate substrates containing canonical RxL motifs. Finally, we investigated whether Cdc13-L-Cdc2 is predicted to preferentially bind DNA replication substrates over mitotic substrates but found no evidence of differential binding. These results reveal diversity in cyclin-CDK substrate recognition beyond established docking motifs.

Heterochromatin protein 1 (HP1 is an intrinsic component of heterochromatin domains where it is involved in a diverse set of functions including heterochromatin spreading and organization, chromatin compaction and transcriptional silencing. It has been suggested that HP1 functions through a phase separation mechanism, a process that has been observed in vitro in the presence of N-terminal phosphorylation, nucleic acids and nucleosome arrays. HP1 can also interact with numerous binding partners that contain a specific motif called an HP1 access code (HAC). HACs recognize and bind to an interface formed by the chromoshadow (CSD) domains in the HP1 homodimer, the functional form of the protein. It has been shown that some HP1 binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1 and three binding partners, namely the p150 subunit of the chromatin assembly factor 1 (CAF-1), the N-terminal domain of the lamin B receptor (LBR), and the mitotic protein Shugoshin 1 (Sgo1). Using phase separation assays, we show that CAF-1 prevents HP1 phase separation while LBR and Sgo1 enhance it. Binding assays, mutational studies, NMR spectroscopy and computational analysis allow us to dissect the contributions of the HAC motifs, the charge patterns of the binding partner sequences and the role of N-terminal phosphorylation on HP1 in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1, while electrostatic interactions dominate the regulation of phosphorylated HP1. These results suggest that HP1 binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts.

Parental care can have pervasive effects on offsprings neurodevelopment. Parent-offspring interactions are often modulated by the neuropeptide oxytocin, which is responsible for the development of social bonds. The development of the oxytocinergic system is dependent on the quality of parental care during the post-natal phase. However, it is yet unknown how post-natal direct interactions can influence the development of the oxytocinergic pathway. Here we tested how an obligate parental care behaviour, tadpole transport in poison frogs, influences the development of the oxytocinergic pathway. To this end, we quantified whole brain expression of oxytocin receptor and oxytocin precursor throughout three developmental stages of A. femoralis tadpoles, before, during and after tadpole transport. Our results show an overall downregulation during tadpole transport, which indicates that during transport tadpoles enter a dormant state to slow down development until they are placed in water. Interestingly, the expression of oxytocin precursor did not vary between the three developmental stages. This might indicate that oxytocin is being recruited during transport, but does not lead to neurodevelopmental changes. In sum, here we present the first evidence of a dormant state during tadpole transport which might be an adaptive response to the terrestrial reproduction in poison frogs.

EIF2alpha kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2alpha-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-delta-KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-delta-KI and of its complex with ATP at 2.1 & 2.5 Angstrom resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-delta-KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

Defects in lysosomal lipid handling provoke fatal disorders presenting neurovisceral symptoms with variable onset and life spans. A prime example is Niemann-Pick type C disease (NPCD), where export of cholesterol and other lipids from the endosomal-lysosomal system is impaired due to variants of either NPC intracellular cholesterol transporter 1 (NPC1) or NPC intracellular cholesterol transporter 2 (NPC2). Therapeutic options for NPCD are limited to palliative care and disease-modifying drugs, and there is an unmet need for new treatments. Based on positive effects in patient-derived fibroblasts in vitro, we explored how inhibition of bromodomain and extra-terminal domain (BET) proteins affects a well-established mouse model bearing the frequent I1061T variant of NPC1. Treatment with JQ1, a hydrophobic prototype BET protein inhibitor, induced beneficial but sex-dependent molecular and behavioral changes in mice. Our results indicate bromodomain proteins as therapeutic drug target for NPCD and reveal sex-dependent BET protein signaling in mice.

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.