Mitochondrion

While long-read sequencing technologies (e.g., PacBio Revio, ONT) have revolutionized high-quality genome assembly for the human pangenome, mitochondrial genome (mtDNA) analysis still largely relies on short-read and Sanger sequencing. However, short-read sequencing often lacks the resolution required to resolve complex variations due to the unique features of mtDNA, such as high mutation rates and repetitive homopolymeric regions, which frequently lead to alignment artifacts and mapping ambiguities. To address this, we evaluated whether applying long-read sequencing to mtDNA improves analytical quality in empirical data. Through comprehensive bioinformatics analyses, we compared the performance of long-read sequencing against short-read sequencing and microarrays. Our results revealed that long-read sequencing detected the highest number of variants (n = 533), significantly outperforming both short-read sequencing (n = 525) and microarrays (n = 49). Notably, both sequencing methods provided significantly higher resolution in haplogroup assignment compared to microarrays in terms of phylogenetic depth (p < 0.05). Long-read sequencing demonstrated superior detection power, particularly for InDels. We identified two novel non-synonymous variants, including a unique InDel detected exclusively by long-read sequencing. Protein modeling and stability analysis validated that this InDel causes structural instability (RMSD > 2.0 [A],{Delta}{Delta} G = -45.21 kcal/mol). Furthermore, we confirmed that this novel InDel is shared among haplogroup A samples in both the 1000 Genomes Project ONT dataset and the Korean population, highlighting the practical implications of long-read sequencing for molecular biology and population genetics.

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

Mitochondrial diseases (MDs) are clinically heterogeneous rendering ascertainment challenging. Estimates of pathogenic mitochondrial DNA (mtDNA) variants in the population range from 1 in 200 to 1 in 4,000 individuals. Inclusion of mtDNA sequencing in genomic databases facilitates comprehensive estimation of mtDNA variation. However, interpretation of low heteroplasmy variation is complex, due in part to misalignment of nuclear mitochondrial DNA transcripts (NUMTs), whilst conservative heteroplasmy thresholds likely omit relevant variation. Cumulative burden of mtDNA variation contributes to aging and neurodegeneration, and recent characterisation of mitochondrial genome constraint allows quantitation of this burden. We analysed whole genome sequencing of blood DNA from 3,500 healthy older individuals in the Medical Genome Reference Bank using mity, considering pathogenic mtDNA variants [&ge;]1% heteroplasmy. We identified 34 distinct pathogenic mtDNA variants in 62 individuals, giving a combined population allele frequency of 1.77% (95% CI 1.36-2.27) or 1 in 56 individuals. We evaluated inclusion of false positive (FP) calls due to two common NUMTs, which accounted for up to 16% of variants. Increasing heteroplasmy thresholding to eliminate all NUMT-FPs also eliminated much of the total variation, including pathogenic variants. We propose a sample-specific, scaled heteroplasmy threshold to maximise variant retention and mitigate NUMT-FPs. Finally, we characterised measures of mitochondrial constraint in this healthy older cohort, observing an association between variant burden and summed constraint, whilst mean constraint was higher in pathogenic variant carriers. These findings suggest pathogenic mtDNA variation is more common in the population than is currently appreciated. Findings are comparable to larger genomic databases when heteroplasmy thresholding is adjusted, and support earlier population-based estimates. Incorporation of low heteroplasmy variation is relevant, but interpretation is nuanced, and optimising variant retention requires consideration of NUMT-FP rates.

Genome sequencing of the heterogeneous primary mitochondrial disorders (PMD) frequently reveals variants of uncertain significance that require functional tests for diagnosis, and does not identify variants in all patients. We analyzed mitochondrial enzyme assays, blue native polyacrylamide gel electrophoresis (BN-PAGE) with in-gel activity staining, complex I assembly blot, and select protein abundances in fibroblasts of a case series of 204 PMD patients divided into functional classes, in comparison to 51 controls and 53 differential diagnostic conditions. Overall, sensitivity and specificity for respiratory chain enzyme assays were 46% and 93% respectively, for BN-PAGE 40% and 98%, for complex I assembly assay 49% and 99%. The overall sensitivity of all tests was 76%, specificity 93%, with positive predictive value 96% and negative predictive value 67%. Categories with high sensitivity were isolated complex deficiencies, nuclear DNA-encoded mitochondrial protein synthesis defects, co-factor defects, and mitochondrial amino-acyl-tRNA synthetase conditions when aided by protein abundance. Mitochondrial DNA mutations and maintenance disorders showed poor sensitivities. Secondary dysfunctions were rare. A complete battery of functional tests showed strong diagnostic clinical utility in fibroblasts.

Background: Low-frequency heteroplasmic mitochondrial DNA (mtDNA) variants are associated with aging and neurological diseases, including Parkinson's disease (PD). Targeted deep mtDNA sequencing using PacBio HiFi long reads has the potential to resolve heteroplasmy across the full mitochondrial genome with high accuracy. Methods: To validate Vega PacBio sequencing for detecting mtDNA heteroplasmy, we analyzed four predefined mixtures of two mtDNA haplotypes. We generated a single long-range PCR amplicon covering the entire mitochondrial genome. These amplicons were mixed at predefined ratios (minor mixture haplotype component: 5%, 2%, 1%, and 0.1%). Variant calling was performed using Mutserve2, and accuracy was assessed by calculating the F1 score from comparisons between expected and detected variants. Full-length mtDNA PacBio sequencing was applied to investigate heteroplasmy across fibroblast passages derived from five LRRK2 p.Gly2019Ser variant carriers (n=3 affected with PD and n=2 unaffected carriers). Changes in mtDNA heteroplasmy level and variant load were assessed longitudinally using a linear mixed model. Results: The single-amplicon approach enabled full-length haplotype resolution without amplification bias associated with overlapping PCR strategies. The F1 score of the predefined mixtures was 1.0 for heteroplasmy levels between 5% and 1% and remained high (0.91) at 0.1%. We detected n=10/62 variants discordant with the Illumina reference at the 0.1% mixture, but sensitivity remained very high at 1.00 in that mixture. Detected minor variants closely matched expected heteroplasmy levels, with average variant levels of 0.057 (5%), 0.022 (2%), 0.011 (1%), and 0.001 (0.1%). Across twelve fibroblast passages, we observed fewer mtDNA heteroplasmic variants ({beta}=-3.2, p=0.026). Increased heteroplasmic variant load over time was also associated with older age ({beta}=1.50, p=0.001) and PD affection status ({beta}=5.0, p=1.0 x 10-4) in LRRK2 variant carriers. Notably, we observed distinct patterns of heteroplasmic variants that either increased or decreased in heteroplasmy level across passages. Conclusion: PacBio HiFi sequencing, combined with a single-amplicon strategy, enables accurate full-length mtDNA heteroplasmy detection and longitudinal analysis, providing a valuable tool for studying mitochondrial variation and dynamics in disease.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

The synthesis of mitochondrial-encoded polypeptides is an essential process, primarily regulated at the posttranscriptional level. In yeast, many regulatory factors have been described as acting in proximity to the mitoribosome to promote efficient translation; however, the precise mechanisms by which these components function remain largely unknown. Here, we expand on findings concerning a previously studied mitoribosome interactor, Mrx9, which is found in large expressosome-like assemblies of mitoribosome clusters. Mrx9 was initially linked to mitochondrial translation and was suggested to be associated with the splicing of COX1 and COB transcripts. Our current data show that Mrx9 is associated with the PHB/m-AAA complex at the polypeptide exit tunnel of the mitoribosome. Overexpression of Mrx9 impairs the proteolytic functions of Yta10 and Yta12 within the prohibitin complex, leading to splicing defects; accumulation of aberrant polypeptides; and a noticeable impairment in the processing of the essential mitoribosomal protein bL32m. These findings support a regulatory role for Mrx9 in the PHB/m-AAA complex by modulating the activities of both Yta10 and Yta12.

Despite their central role in metabolite exchange, lipid trafficking, and protein import, chloroplast outer envelope beta-barrel proteins lack a dedicated comprehensive sequence database spanning many plant proteomes. Here we present the database COB (chloroplast outer-envelope beta-barrel), consisting of 16,586 beta-barrel sequences organized across ten protein categories and an uncharacterized group. COB was constructed using a machine learning classifier that identifies chloroplast beta-barrels based on features derived from evolutionary protein contact maps. Analysis of COB reveals that Streptophyta have more barrels overall and use a greater variety of solute transporters than Chlorophyta. Furthermore, we find considerable structural diversity across OEP categories, including variation in beta-strand count and a high prevalence of open barrel conformations not observed in bacterial outer membrane proteins. Structure predictions for Arabidopsis thaliana outer envelope proteins identified candidate hybrid barrel assemblies, with TOC159 family members emerging as universal interaction partners. We also report single-chain multi-barrel domain architectures in the chloroplast outer envelope, a topology previously described only in Gram-negative bacteria. Finally, we find chloroplast membrane barrels have more open topologies and shorter strands than bacterial membrane barrels. COB provides a comprehensive sequence resource for chloroplast outer envelope beta-barrels and establishes a foundation for investigating the evolution, structure, and function of this essential protein in chloroplast.

Mitochondria integrate bioenergetic metabolism, redox control, genome maintenance, and stress signaling across all eukaryotes. Although plant and human mitochondria diverged substantially during evolution, both systems retain systems-level principles for sensing mitochondrial dysfunction and communicating stress signals to the nucleus. Here, we develop an integrative comparative in silico framework to evaluate whether plant mitochondrial stress signaling can provide a useful conceptual model for interpreting human mitochondrial disease vulnerability. Core Arabidopsis thaliana regulators representing alternative respiration, mitochondrial retrograde signaling, translational stress control, and genome surveillance were compared with functionally analogous human regulators involved in integrated stress response (ISR) signaling, mitochondrial DNA maintenance, and mitochondrial disease phenotypes. Domain architecture, protein-protein interaction topology, enrichment profiles, disease-gene associations, and promoter motif architecture were integrated to assess cross-kingdom convergence at the level of stress-response organization rather than direct orthologs. The plant network formed a compact AOX-NAC-centered stress module associated with respiratory flexibility and retrograde signaling, whereas the human network displayed expanded ISR and mtDNA maintenance modules enriched for mitochondrial disease associations. Promoter motif analyses further indicated lineage-specific transcription factor signatures but broadly comparable stress-responsive regulatory logic. Collectively, these results support the concept that plant mitochondrial stress systems represent simplified resilience-oriented architectures that can help generate experimentally testable hypotheses about failure points in human mitochondrial stress responses.

Disorders of mitochondrial oxidative phosphorylation affecting multiple respiratory chain complexes are among the most common causes of mitochondrial disease in humans. However, impaired energy metabolism alone does not fully account for tissue-specific vulnerability and disease progression, suggesting that additional molecular mechanisms contribute to disease pathology. We previously identified PTCD1 variants in a child with infantile cardiomyopathy associated with a combined respiratory chain deficiency. Here, we establish the pathogenicity of three PTCD1 (NM_015545.4) variants in which p.(Arg113Trp) and p.(Gly184Arg) segregate in cis whereas p.(Arg130*) is present in trans, demonstrating that disrupted mitochondrial proteostasis contributes to tissue damage in PTCD1 deficiency. PTCD1 patient cardiac tissue characterisation revealed impaired mitoribosome biogenesis, alongside increased aggregation of selective mitochondrial matrix proteins. Cell models expressing individual and combined PTCD1 missense variants, coupled with proteomics, recapitulated the protein aggregation, with the cis p.(Arg113Trp);p.(Gly184Arg) combination showing the most severe effect. Protein aggregation was accompanied by altered OPA1 processing and mitochondrial network remodelling. Our findings establish accumulating proteotoxic stress arising from impaired mitoribosome assembly as a pathogenic mechanism in post-mitotic tissues, driving PTCD1 cardiomyopathy.

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

The existence and functional relevance of mitochondrial DNA methylation remain controversial. Here, we systematically profiled cytosine methylation and hydroxymethylation across human brain and blood tissues spanning healthy and malignant states using orthogonal sequencing approaches that avoid chemical conversion during library preparation. While nuclear DNA exhibited canonical methylation patterns, mitochondrial DNA consistently showed negligible signal, indistinguishable from background technical noise. By mapping cytosine-guanine sites between mitochondrial DNA and nuclear-embedded mitochondrial sequences, we demonstrate the potential of these nuclear counterparts to confound not only cytosine methylation but also hydroxymethylation measurements, corroborating and extending prior findings implicating nuclear contamination as a potential source of apparent mitochondrial epigenetic signals. Additional technical factors that inflate apparent mtDNA methylation signals were identified, including sequence context biases, flow cell chemistries, and coverage-dependent discrepancies between the heavy and light strands. Collectively, these results provide convergent evidence against the presence of biologically meaningful cytosine methylation or hydroxymethylation in mitochondrial DNA. These findings caution against interpreting apparent mtDNA methylation signals in human adult tissues as meaningful without rigorous orthogonal validation and comprehensive consideration of technical and analytical confounding factors.

The cofactor Fe-protoporphyrin IX cofactor (heme) performs many functions in biology. Animal cells must stabilize their newly generated heme-free (apo)-hemeproteins and deliver mitochondrial heme to them so they can mature to functional form. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) typically accomplishes the heme deliveries, and for many apo-hemeproteins, heat shock protein 90 (Hsp90) drives their heme insertions. We previously observed hemeproteins express poorly in a cell line (COS-7) that does not express soluble guanylyl cyclase (sGC), a heme-binding enzyme that typically functions through its cGMP generation. To understand sGC involvement, we expressed four hemeproteins, Hemoglobin beta (Hb{beta}), Myoglobin (Mb), Indoleamine 2,3-dioxygenase 1 (IDO1), and Tryptophan 2,3-dioxygenase (TDO) in a cell line expressing sGC (HEK293) or in two cell lines (COS-7, DU145) that do not. We assessed hemeprotein expression levels, their abilities to acquire heme, and when relevant if these facets could be rescued by co-expressing individual sGC subunits, including variants with defects in either sGC heme binding, Hsp90 association, heterodimerization, or cGMP production. We found that co-expression of either sGC subunit was essential for three of the four apo-hemeproteins to accumulate in the COS7 and DU145 cells and acquire heme. This did not involve heme binding, heterodimer formation, or cGMP generation by the sGC subunits, and instead depended on a subunits ability to recruit Hsp90 and GAPDH to the apo-hemeproteins via their own Hsp90 binding. Recruiting Hsp90 and GAPDH to apo-hemeprotein clients to ensure they can accumulate and mature to functional form broadens our understanding of sGC and Hsp90 functions in biology.

We show that chronic impairment of mitochondrial respiration is associated with marked accumulation of cytochrome c (Cytc) protein. Using SCO2-deficient HCT116 cells lacking functional cytochrome c oxidase and wild-type cells exposed to sustained hypoxia, we found that substantial mitochondrial Cytc accumulation parallels reduced electron flux through Cytc. SCO2-deficient cells exhibited equally elevated Cytc levels under normoxia (19% O2) and hypoxia (0.1-3% O2). Wild-type cells under sustained hypoxia accumulated Cytc, reaching levels comparable to those in SCO2-deficient cells. This effect was reversible upon reoxygenation. Increased Cytc protein levels were also observed in other cell models, including primary cortical neurons cultured under chronic hypoxia and in cerebral cortex tissue from hypoxia-exposed mice. Cytc accumulation occurred independently of CYCS transcription, mRNA translation, HIF activation, ROS production and changes in mitochondrial network. Pharmacological inhibition of complex III was likewise accompanied by increased Cytc levels, whereas mitochondrial uncoupling had no effect, suggesting that impaired electron transfer rather than membrane depolarisation per se underlies this association. Raman spectroscopy revealed enrichment of reduced Cytc and an increased Cytc-to-cytochrome b ratio in respiration-deficient cells. Further supporting a stabilisation-based mechanism, the fraction of membrane-unbound ferro-Cytc was decreased in SCO2-deficient cells, consistent with moderate cardiolipin enrichment, which is known to enhance retention of Cytc at the inner mitochondrial membrane. Despite elevated mitochondrial Cytc content, SCO2-deficient cells were less susceptible to apoptosis induced by intermittent hypoxia or dichloroacetate. Together, these findings indicate that reduced electron flux through complex IV is associated with Cytc accumulation through increased protein stability and membrane retention without enhancing apoptotic sensitivity.

Language genes can be tentatively considered as a subset of cognitive genes, although they are often discussed separately. During the evolution of SNVs (single nucleotide variations) in cognition-related genes, do language genes and cognitive genes exhibit significantly different intensities of change at several key evolutionary moments--namely, the inflection points or derivative peak positions of similarity curves drawn from multi-SNV locus bases across samples? In this study, nine distance/similarity metrics (Bray-Curtis, Cosine, Pearson, Spearman, Hamming, Jaccard, Matching, Kulczynski, and Gower) were employed to analyze 413 samples from 11 taxonomic groups, targeting SNV loci in language/cognition-related genes (13,415 effective loci, approximately 400 loci per gene), with pp6 (Homo_sapiens.GRCh38) as the reference. For each method, sample similarities (defined as 1/(1+distance)) were independently sorted in ascending order to generate raw similarity scatterplots. Due to the large sample size and representativeness, the scatter density on the similarity curves was high, and no smoothing was applied. Derivative values were calculated from adjacent similarity differences to identify peaks of evolutionary rate change (top 10 peaks per method). Combined with functional annotations of 33 language/cognition-related genes, we quantified the difference scores and occurrence frequencies of the two gene categories at the peak positions. The results indicate that cognitive-related genes exhibit slightly higher occurrence frequencies in peak windows and higher average difference scores per gene than language genes. Comparative analysis of SNVs at the peak samples and their left-side windows revealed that at positions 381-382, all nine methods shared three intersecting mutation loci, involving language genes (NFXL1, SRGAP2, SRGAP2C); at positions 355-356, there was one intersecting mutation locus, involving a language gene (SRGAP2). This suggests that certain mutations in language genes may have played a distinctive role at critical junctures in the evolution of cognitive abilities.

Primary mitochondrial diseases (PMD) have limited disease-modifying therapies, currently applicable to only 3 of over 400 discrete gene disorders. Cycloheximide (CHX) is a global cytosolic translation inhibitor we previously reported to rescue PMD preclinical models, although its toxicity precluded clinical development. To identify specific mediators underlying CHX treatment benefit in PMD, SOMAscan-based proteomics was performed in complex I deficient and genetic disease fibroblast cell line models grown in galactose. Thrombopoietin (THPO) and insulin-like growth factor binding protein 5 (IGFBP5) were the only two differentially regulated proteins, together with ERK/MAPK pathway dysregulation, identified upon CHX treatment in PMD versus healthy control cells. THPO inhibition by siRNA or pharmacologic approaches rescued stress-induced viability loss in patient fibroblasts having diverse PMD gene etiologies, and significantly improved mitochondrial stress, linear growth, and neuromuscular function in a classical ndufs2-/- C. elegans model. IGFBP5 overexpression by lentiviral or mRNA approaches rescued cell viability across distinct PMD gene etiologies, as did IGF1 pharmacologic inhibition across both PMD mutant and C. elegans models. MAPK pharmacologic inhibition rescued multiple distinct complex I disease cells survival, as well as mitochondrial stress in SLC25A46-/- C. elegans. Combination therapies targeting multiple of these glucose signaling pathway proteins, together with glucose and N-acetylcysteine, yielded superior therapeutic benefit in complex I disease cell and C. elegans models. Additionally, single or combined pharmacologic inhibition of THPO or IGF1 significantly enhanced primary and metastatic osteosarcoma cell death. Collectively, targeted small molecule and genetic modulation of THPO, IGF1, or MAPK recapitulated the significant therapeutic benefit of CHX in PMD, while avoiding global translation inhibition. These novel PMD therapies likely confer benefit by attenuating MAPK-driven autophagy and potentially promoting noncanonical glucose uptake, improving cellular energy balance. Overall, these glucose signaling cellular pathway targets hold broad therapeutic promise for PMD patients, warranting further clinical research development.

RationaleRecent viral metagenomic studies have identified a plethora of enzyme-encoding genes in Phycodnaviridae viruses that are not strictly required for viral replication. These enzymes hold an unexpected metabolic potential during the infection process with their specific green algae host. As neither their role in the infection process nor the subcellular localization of these proteins has been experimentally characterized, comparative sequences, structural and biochemical in silico analyses can help generate functional and localization hypotheses. MethodsIn a recent viral metagenomic dataset, we identified a collection of viral homologs involved in bilin biosynthesis: heme oxygenase (vHMOX1) and Phycocyanobilin:Ferredoxin oxidoreductase (vPcyA). Viral and algal homologues were compared through sequence analyses and AlphaFold3 structural predictions. Predicted biochemical properties were analyzed for their compatibility with subcellular compartments. Active site architecture and putative substrate binding were compared between viral and algal proteins using AlphaFold3 and experimentally resolved structures. ResultsViral HMOX1 and PcyA sequences are truncated compared to algal homologs, lacking the N-terminal extension associated with chloroplast targeting. However biochemical properties, including isoelectric point and surface charge distribution, are compatible with localization in chloroplast stroma. Structural comparisons reveal modifications in the viral HMOX1 active site, including partial substrate reorientation and substitutions of key residues, consistent with modified heme-binding properties. In contrast, vPcyA models show no significant differences to their algal counterparts. ConclusionsActive site remodeling in vHMOX1 protein models suggests that these viral homologues may have evolved distinct heme-binding properties. Unlike vPcyA, vHMOX1 homologs appear to have diverged more substantially from their algal counterparts, potentially reflecting functional specialization in the viral infection context. One sentence summary of key findingsOur bioinformatic analyses expand the repertoire of auxiliary metabolic genes in Phycodnaviridae by identifying a conserved heme degradation pathway, non-canonical vHMOX1/PcyA targeting and structural rearrangements surrounding the catalytic sites of viral HMOX1.

Horizontal gene transfer (HGT) drives organellar evolution, particularly in parasitic plants where host connections facilitate extensive DNA exchange. However, how these processes intersect with cellular machinery to reshape mitogenomic architecture remains poorly understood. Here, we investigate the mechanisms governing structural plasticity and asymmetric host-DNA integration in the extreme holoparasitic family Rafflesiaceae. By performing a comprehensive comparative analysis across all three extant genera (Sapria, Rhizanthes, and Rafflesia) and their Tetrastigma host lineage, we discovered extraordinary mitogenome size divergence, ranging from the expanded 824-kb genome of Sapria (40 circular chromosomes) to the streamlined 282-kb genome of Rhizanthes (35 circular chromosomes). Strikingly, these closely related genera display a total lack of chromosomal synteny, which we link to the ancestral loss of key recombination surveillance genes (RECX, ODB1). Furthermore, while all three genera strictly conserve an identical core of 30 protein-coding genes, host-derived HGT is highly asymmetric, ranging from minimal in Rhizanthes to 60% in Sapria. In Sapria, foreign tracts are sequestered into 15 predominantly non-coding circular chromosomes, a structural arrangement that aligns with the circle-mediated HGT model validated in other holoparasites. Collectively, these parallel patterns across phylogenetically distant lineages demonstrate that sorting and maintaining foreign DNA in autonomous circular blocks is a convergent architectural outcome of massive host-to-parasite genetic transfers. SIGNIFICANCE STATEMENTHorizontal gene transfer is widespread in the nuclear genome of the parasitic plant family Rafflesiaceae, but its contribution to mitochondrial genome evolution has been assessed through the analyses of a limited number of genes. By comparing complete mitochondrial genomes of the parasites and their hosts, we found that closely related species evolved dramatically different genome architectures through distinct mechanisms: one lineage accumulated large amounts of host-derived DNA, whereas another expanded through the proliferation of repetitive sequences with limited contribution from foreign DNA. These findings show that different evolutionary processes can generate profoundly divergent mitochondrial genomes even among closely related parasitic plants.

Mitochondria are cellular energy hubs best known for ATP production via oxidative phosphorylation; however, they also serve as biosynthetic centers for phospholipids. Mitochondrial phospholipids are critical for various cellular processes, and their loss underlies myriad mitochondrial diseases. The critical enzymes underlying these biosynthetic cascades are encoded in the nucleus, translated in the cytosol, and imported into mitochondria. Understanding of mechanisms and factors that ensure precise targeting of proteins to mitochondria has been long overlooked but remains critical. Recently, the J-protein/Hsp40 cochaperone Djp1 has emerged as a key player in mitochondrial protein targeting by promoting the transfer of precursors from the endoplasmic reticulum (ER) surface to mitochondria in a pathway termed ER-SURF. Molecular details regarding how Djp1 recognizes clients and more broadly supports mitochondrial function remain unknown. Using biochemical approaches, proteomics, and thin layer chromatography, we demonstrate that Djp1 is a regulator of Phosphatidylserine decarboxylase 1 (Psd1), an inner mitochondrial membrane resident responsible for mitochondrial phosphatidylethanolamine (PE) production. This regulation of Psd1 biogenesis is dependent on its mitochondrial targeting signal and is specific to Djp1 compared to other members of the Hsp40 family or ER targeting factors. Intriguingly, the combined loss of Djp1 and Psd1 results in a synthetic sick phenotype that unexpectedly reflects a role(s) for Djp1 in proper mitochondrial phospholipid metabolism independent of Psd1. Taken together, these findings expand our understanding of Djp1-dependent mitochondrial protein regulation and unveil Djp1 as important for mitochondrial phospholipid metabolism by multiple mechanisms.

Bi-allelic mutations in MSTO1 are linked to clinical disease phenotypes characteristic of mitochondrial dysfunction, including ataxia and muscular dystrophy. Consistent with this, MSTO1 patient-derived fibroblasts have fragmented mitochondria and a striking loss of mtDNA. Although MSTO1 has been implicated in regulating mitochondrial fusion, the molecular function of this cytosolic protein in vertebrate cells remains unclear. Using the auxin-inducible degradation (AID) system we demonstrate that MSTO1-FLAG-AID protein is rapidly depleted to almost undetectable levels. Importantly, these cells recapitulate the fragmented mitochondrial phenotype observed in patients and thus are a valuable model of disease. Surprisingly, prior to any changes in mitochondria, we show that MSTO1-depleted cells have a significant decrease in TRiC levels, an essential cytosolic ATP-dependent chaperone required to fold diverse substrates, including actin and tubulin. We reveal that TRiC is also reduced in MSTO1 patient-derived fibroblasts, indicating that loss of TRiC may contribute to disease pathophysiology. We further demonstrate that knockdown of TRiC leads to a decrease in MSTO1 protein levels and remarkably, was sufficient to induce a fragmented mitochondrial phenotype, independent of changes in tubulin or actin. This reveals a previously unrecognized connection between TRiC and mitochondrial homeostasis. Using co-immunoprecipitation we found that MSTO1 interacts with the TRiC chaperone. We also observe accumulation of early TRiC assembly subcomplexes in the absence of MSTO1 suggesting that MSTO1 facilitates assembly of TRiC. Together, our findings identify MSTO1 as a TRiC assembly factor and connect mitochondrial defects caused by MSTO1-depletion to the loss of TRiC. SignificanceMSTO1, a protein linked to myopathy and ataxia, has been thought to control mitochondrial fusion, although the molecular mechanism is unknown. Using rapid depletion of MSTO1, we found that mitochondrial fragmentation appears only after six days. Significantly, the levels of the essential cytosolic chaperonin TRiC are reduced within two days of MSTO1 depletion. Directly depleting TRiC reproduces the fragmented mitochondrial phenotype seen with loss of MSTO1, consistent with a model where mitochondrial dysfunction is a downstream consequence of impaired protein folding rather than a direct effect of MSTO1 loss. We show that MSTO1 is required for assembly of TRiC, identifying it as a long-sought assembly factor for this macromolecular protein complex.