Recombinant Schizosaccharomyces pombe ATP synthase subunit J, mitochondrial (atp18)

What is ATP synthase subunit J in S. pombe and what is its role in mitochondrial function?

ATP synthase subunit J (atp18) is a small protein component of the mitochondrial F1F0-ATP synthase complex in Schizosaccharomyces pombe. It functions as part of the membrane-embedded F0 sector, which is responsible for proton translocation across the inner mitochondrial membrane. In S. pombe, as in other eukaryotes, the ATP synthase complex utilizes the proton gradient generated by the electron transport chain to synthesize ATP from ADP and inorganic phosphate. The subunit J specifically contributes to the structural stability of the F0 sector and may play a role in the oligomerization of ATP synthase complexes into dimers and higher-order structures, which are important for cristae formation in the inner mitochondrial membrane. While studies on ATP synthase in S. pombe have primarily focused on the major subunits, comparative analyses with other yeast species suggest that subunit J contributes to the efficiency of ATP production and mitochondrial morphology maintenance .

What experimental approaches are recommended for confirming subcellular localization of ATP synthase subunit J?

To confirm the mitochondrial localization of ATP synthase subunit J in S. pombe, a multi-method approach is recommended. Fluorescence microscopy using GFP-tagged atp18 provides a straightforward visualization method, building on S. pombe's established protocols for protein localization as demonstrated in the 2006 proteome-wide localization studies . For higher resolution analysis, immunogold electron microscopy offers precise localization within mitochondrial compartments. Biochemical fractionation techniques should be employed to isolate mitochondria, followed by Western blotting with antibodies against atp18 and mitochondrial markers. Researchers should use established protocols for S. pombe subcellular fractionation, which differ from those used for S. cerevisiae due to differences in cell wall composition. For quantitative assessment, researchers can perform co-localization studies with known mitochondrial proteins such as components of respiratory complexes. Additionally, importing in vitro translated radiolabeled atp18 into isolated mitochondria can confirm the protein's mitochondrial targeting sequence functionality and import pathway. Each method offers complementary information, and convergent results from multiple approaches provide the strongest evidence for mitochondrial localization.

What phenotypes are associated with atp18 deletions or mutations in S. pombe?

Deletion or mutation of atp18 in S. pombe typically results in respiratory deficiency phenotypes, though the severity depends on the specific mutation and genetic background. Complete deletion often leads to petite colony formation on fermentable carbon sources and failure to grow on non-fermentable carbon sources such as glycerol or ethanol, indicating compromised oxidative phosphorylation. At the cellular level, atp18 mutants frequently exhibit abnormal mitochondrial morphology, including fragmentation and cristae disorganization, visible by electron microscopy. ATP production measurements in isolated mitochondria from these mutants show reduced ATP synthesis rates compared to wild-type strains. Point mutations in conserved residues of atp18 can produce partial loss-of-function phenotypes, with temperature-sensitive growth defects similar to those observed in other S. pombe mitochondrial protein mutants . Studies of S. pombe mutants with modified alpha subunits of mitochondrial ATPase have shown altered enzymatic properties, including changes in ADP sensitivity and cooperativity patterns . These observations suggest that mutations in other subunits like atp18 could similarly alter ATP synthase function through structural perturbations of the complex.

How do researchers optimize expression and purification of recombinant S. pombe ATP synthase subunit J?

Optimizing expression and purification of recombinant S. pombe ATP synthase subunit J requires strategic consideration of expression systems and purification techniques. For bacterial expression, E. coli BL21(DE3) strains with pET-based vectors provide high yield, though expression of this mitochondrial membrane protein often leads to inclusion bodies. Expression should be induced at lower temperatures (16-18°C) with reduced IPTG concentrations (0.1-0.3 mM) to enhance proper folding. For native conformation, expression in yeast systems is preferred, with S. pombe itself serving as an excellent host using vectors like pREP series with thiamine-repressible promoters.

For purification from S. pombe, a protocol similar to that used for the Rad18 complex can be adapted . Cells should be grown to mid-log phase, harvested, and lysed in buffer containing 45 mM HEPES (pH 7.8), 300 mM KCl, 10% glycerol, protease inhibitors, and detergents suitable for membrane proteins (e.g., 1% digitonin or 0.5% DDM). After centrifugation to remove cell debris, mitochondrial membranes can be isolated by differential centrifugation. Solubilization of ATP synthase complexes requires gentle detergents to maintain native interactions.

Affinity purification using N- or C-terminal tags (His6 or FLAG) provides the first purification step, followed by ion exchange chromatography and size exclusion chromatography. For structural studies, additional steps such as gradient ultracentrifugation may be necessary to isolate intact ATP synthase complexes containing subunit J. Protein purity should be assessed by SDS-PAGE with silver staining, and identity confirmed by mass spectrometry.

What approaches are most effective for studying interactions between ATP synthase subunit J and other complex components?

For studying interactions between ATP synthase subunit J and other complex components in S. pombe, researchers should employ a combination of in vivo and in vitro techniques. Co-immunoprecipitation (Co-IP) provides direct evidence of protein-protein interactions within native complexes. This approach can utilize antibodies against tagged versions of atp18 or other ATP synthase components, following protocols similar to those used for studying the Smc5-6 complex in S. pombe .

Crosslinking mass spectrometry (XL-MS) offers valuable insights into the spatial relationships between subunits. Chemical crosslinkers of various lengths can be used to capture transient or stable interactions, followed by digestion and MS analysis to identify crosslinked peptides. For S. pombe ATP synthase, a crosslinking approach with MS-compatible reagents like DSS or EDC is recommended, with appropriate optimization of crosslinking conditions to prevent non-specific interactions.

Yeast two-hybrid (Y2H) assays can identify binary interactions, though membrane proteins like atp18 may require modification (using soluble domains) for effective analysis. Split-ubiquitin membrane Y2H systems are more appropriate for full-length membrane proteins. Fluorescence resonance energy transfer (FRET) analysis using fluorescently tagged proteins provides spatial resolution of interactions in living cells.

In vitro approaches include reconstitution experiments using purified components. For subunit J specifically, in vitro transcription-translation systems similar to those used for Rad18-Spr18 interaction studies can be adapted, expressing tagged versions of atp18 and potential interacting partners for subsequent co-purification analysis.

How do mutations in the atp18 gene affect ATP synthase activity and oligomerization?

Mutations in the atp18 gene can substantially affect both ATP synthase catalytic activity and supramolecular organization. Point mutations in conserved residues often lead to reduced ATP synthesis rates, measurable in isolated mitochondria using luciferase-based ATP detection assays. Structure-function studies indicate that mutations in transmembrane domains typically disrupt complex assembly, while mutations at subunit interfaces may permit assembly but alter enzymatic properties.

Similar to observations with alpha subunit mutants in S. pombe ATP synthase , atp18 mutations can affect regulatory properties such as ADP inhibition sensitivity, proton conductance coupling, and catalytic cooperativity. Specifically, mutations at protein-protein interfaces may alter communication between F1 and F0 sectors, affecting how proton translocation couples to conformational changes required for ATP synthesis.

For oligomerization effects, blue native PAGE analysis of digitonin-solubilized mitochondrial extracts from atp18 mutants typically reveals shifts from dimeric and higher-order oligomeric forms toward monomeric complexes. This reorganization has significant implications for cristae morphology, as ATP synthase dimers contribute to membrane curvature at cristae ridges. Electron microscopy of mitochondria from atp18 mutants often shows altered cristae architecture, with abnormal branching patterns and reduced membrane potential measured by potential-sensitive dyes.

The relationship between oligomerization defects and catalytic efficiency suggests that proper supramolecular organization optimizes proton utilization efficiency. Kinetic analyses of ATP synthase activity in reconstituted proteoliposomes containing wild-type versus mutant complexes can distinguish between effects on intrinsic catalytic activity and those resulting from altered oligomeric states.

What are the evolutionary implications of structural variations in ATP synthase subunit J across species?

Evolutionary analysis of ATP synthase subunit J reveals important structural adaptations across different taxonomic groups, with significant implications for understanding mitochondrial function. While core ATP synthase subunits display high conservation across eukaryotes, smaller subunits like subunit J show greater sequence divergence while maintaining structural and functional roles. In S. pombe, ATP synthase subunit J represents an intermediate evolutionary stage between those found in other fungi and metazoans.

Phylogenetic analyses indicate that ATP synthase subunit J in S. pombe shares approximately 30-35% sequence identity with homologs in S. cerevisiae, but interestingly, certain structural motifs more closely resemble those in mammalian homologs. This evolutionary pattern aligns with other observations showing that S. pombe often serves as a better model for mammalian processes than S. cerevisiae . The transmembrane domains of subunit J show higher conservation than soluble regions, reflecting evolutionary constraints on membrane-embedded structural elements.

Comparative structural modeling suggests that variations in ATP synthase subunit J contribute to species-specific differences in ATP synthase oligomerization and cristae morphology. These adaptations may reflect metabolic demands specific to different organisms' ecological niches. For instance, S. pombe, as a facultative anaerobe capable of growth in varying oxygen conditions, may have evolved specific features in ATP synthase subunit J that optimize performance across different metabolic states .

The evolutionary conservation pattern of ATP synthase subunit J provides insights into essential functional domains versus regions that permit variation. This information guides site-directed mutagenesis approaches for structure-function studies and helps identify residues likely responsible for species-specific properties of mitochondrial ATP production systems.

What are the recommended approaches for generating recombinant S. pombe strains with modified atp18?

For generating recombinant S. pombe strains with modified atp18, researchers should consider several complementary genetic engineering approaches. CRISPR-Cas9 has emerged as the most efficient method for targeted modifications, using protocols adapted specifically for S. pombe's unique cell biology. Researchers should design guide RNAs targeting the atp18 locus with minimal off-target potential, and provide repair templates containing the desired modifications flanked by 500-1000 bp homology arms. Transformation efficiency can be optimized using lithium acetate/PEG with heat shock, followed by selection on appropriate media.

For epitope tagging, both N- and C-terminal tags have been successfully applied to mitochondrial proteins in S. pombe. When working with atp18, C-terminal tagging is generally preferred as N-terminal modifications may interfere with mitochondrial targeting sequences. The PCR-based gene targeting method using the pFA6a plasmid series allows integration of tags such as GFP, HA, or TAP with appropriate selectable markers. This approach was successfully used for proteome-wide localization studies in S. pombe .

For controlled expression studies, the thiamine-repressible nmt1 promoter system provides titratable expression levels through three promoter strengths (nmt1, nmt41, and nmt81). This system is particularly valuable for studying dosage effects of atp18 expression on ATP synthase assembly and function. Site-directed mutagenesis of atp18 can be performed using either base editors with CRISPR-Cas9 or traditional homologous recombination approaches. Following genetic modification, verification should include both genomic PCR and sequencing, as well as Western blot confirmation of protein expression.

How should researchers design experiments to measure ATP synthase activity in S. pombe mitochondria?

Designing experiments to measure ATP synthase activity in S. pombe mitochondria requires careful consideration of isolation methods and activity assays. For mitochondrial isolation, researchers should optimize protocols specifically for S. pombe, which differs from S. cerevisiae in cell wall composition. Enzymatic digestion with Zymolyase-20T followed by gentle mechanical disruption preserves mitochondrial integrity. Isolated mitochondria should be assessed for respiratory control ratios to confirm functional integrity before activity measurements.

ATP synthase activity can be measured through multiple complementary approaches:

• ATP synthesis assay: Measure ATP production rate in freshly isolated mitochondria supplied with ADP, Pi, and respiratory substrates (e.g., succinate with rotenone). ATP production is quantified using a luciferase-based luminescence assay, with calibration curves generated using ATP standards. Oligomycin sensitivity confirms ATP synthase-specific activity.

• ATP hydrolysis assay: Measure the reverse reaction (ATP hydrolysis) spectrophotometrically by coupling ADP production to NADH oxidation through pyruvate kinase and lactate dehydrogenase enzymes. This approach allows kinetic analysis of parameters such as Vm and Km for ATP, similar to studies performed with revertant S. pombe strains .

• Proton pumping assay: Assess proton translocation using fluorescent probes such as ACMA (9-amino-6-chloro-2-methoxyacridine) that quench in response to ΔpH formation.

• Membrane potential measurements: Evaluate mitochondrial membrane potential using potentiometric dyes like TMRM or JC-1, which provide indirect measures of ATP synthase function.

Researchers should include appropriate controls, including measurements in the presence of specific inhibitors (oligomycin for ATP synthase, antimycin A for complex III) to distinguish ATP synthase-specific activities from other processes. For kinetic analysis, researchers can determine parameters such as ADP sensitivity and bicarbonate effects on negative cooperativity, as observed in studies of S. pombe F1 ATPase .

What methods are most effective for analyzing the assembly of ATP synthase complexes containing modified subunit J?

Analyzing ATP synthase complex assembly with modified subunit J requires techniques that preserve native protein interactions while providing high resolution. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) serves as the foundation for assembly analysis, allowing separation of intact ATP synthase complexes. Mitochondria should be solubilized with mild detergents (digitonin at 2-4 g/g protein is recommended) to maintain supramolecular assemblies. For enhanced resolution of assembly intermediates, two-dimensional BN/SDS-PAGE provides visualization of individual subunits within complexes.

Pulse-chase labeling with radioactive amino acids, followed by immunoprecipitation with antibodies against ATP synthase subunits, allows time-course analysis of complex assembly. This approach can reveal whether modified subunit J affects assembly kinetics or stability of the complete complex. For S. pombe specifically, protocols similar to those used for studying the Rad18-Spr18 complex can be adapted, including immunoprecipitation and in vitro transcription-translation methods .

Sucrose gradient ultracentrifugation provides another approach for separating assembly intermediates based on size and density. Fractions collected from gradients can be analyzed by Western blotting to determine the distribution of atp18 and other ATP synthase subunits. Quantitative mass spectrometry using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) can compare the stoichiometry of subunits in assembled complexes between wild-type and modified atp18 strains.

How can researchers effectively use S. pombe as a model for studying human mitochondrial disorders related to ATP synthase defects?

S. pombe offers several advantages as a model for studying human mitochondrial disorders related to ATP synthase defects. To effectively utilize this system, researchers should first identify human ATP synthase mutations of interest and create corresponding mutations in S. pombe homologs. Sequence alignment tools can identify conserved residues between human and S. pombe ATP synthase subunits, enabling targeted mutagenesis of equivalent positions. Once mutations are introduced through CRISPR-Cas9 or homologous recombination approaches, phenotypic characterization should proceed systematically.

Growth assays on fermentable versus non-fermentable carbon sources provide initial assessment of respiratory competence. Measurement of oxygen consumption rates, ATP synthesis capacity, and membrane potential in isolated mitochondria establishes the biochemical consequences of mutations. For morphological analysis, electron microscopy of mitochondrial ultrastructure can reveal cristae abnormalities characteristic of ATP synthase disorders.

S. pombe provides advantages over S. cerevisiae for modeling human disorders due to its more similar mitochondrial genome organization and gene expression mechanisms . Additionally, S. pombe's cell cycle control and checkpoint systems more closely resemble those of humans, allowing better modeling of how energy deficits affect cell cycle progression.

For complementation studies, researchers can express human ATP synthase subunits in S. pombe mutants to test functional conservation. This approach can validate pathogenicity of novel variants identified in patients with suspected mitochondrial disorders. Transcriptomic and proteomic analyses of ATP synthase mutants can identify compensatory responses and secondary effects of ATP synthase dysfunction, potentially revealing therapeutic targets.

The genetic tractability of S. pombe facilitates modifier screens to identify suppressors or enhancers of ATP synthase defects, potentially uncovering novel therapeutic approaches for human mitochondrial disorders.

How can researchers resolve discrepancies in ATP synthase activity measurements from different experimental approaches?

Resolving discrepancies in ATP synthase activity measurements requires systematic analysis of methodological variations and biological factors. When faced with contradictory results, researchers should first examine differences in mitochondrial isolation procedures. The integrity of isolated mitochondria significantly impacts activity measurements; respiratory control ratios should be determined to confirm mitochondrial functionality before ATP synthase assays. Contamination with vacuolar ATPases or plasma membrane ATPases can produce misleading results, necessitating proper fractionation verification.

Assay conditions represent another source of variability. The presence of different ions can substantially alter ATP synthase behavior, as demonstrated in studies of S. pombe F1 ATPase where bicarbonate affects negative cooperativity . Researchers should standardize buffer compositions, substrate concentrations, pH, and temperature across experiments. When comparing ATP synthesis versus hydrolysis activities, remember these reactions may be differentially affected by mutations due to the asymmetric nature of ATP synthase function.

Different detection methods (spectrophotometric, luminescence-based, radiochemical) have varying sensitivities and potential artifacts. Cross-validation using multiple detection methods strengthens confidence in results. When comparing whole-cell versus isolated mitochondria measurements, consider that cellular regulatory mechanisms absent in isolated organelles may influence activity. Similarly, detergent-solubilized enzyme may behave differently than membrane-embedded complexes.

Statistical analysis should account for biological variability by including sufficient biological replicates (minimum n=3) and technical replicates. Analysis of variance (ANOVA) with appropriate post-hoc tests can determine significant differences between conditions. Finally, researchers should consider strain background effects, as secondary mutations may compensate for primary defects in long-established laboratory strains.

What are the common pitfalls in purifying recombinant ATP synthase components from S. pombe and how can they be addressed?

Purification of recombinant ATP synthase components from S. pombe presents several challenges requiring specific troubleshooting approaches. Protein solubility represents a primary hurdle, as hydrophobic membrane proteins like ATP synthase subunit J tend to aggregate during extraction. This can be addressed by optimizing detergent selection—digitonin preserves native interactions but offers limited solubilization, while DDM provides better solubilization but may disrupt some protein-protein interactions. A staged approach using different detergents for extraction versus purification steps often yields better results.

Low expression levels frequently limit yield. To overcome this, researchers should compare different promoter systems; the nmt1 promoter series offers titratable expression levels in S. pombe. Codon optimization for highly expressed S. pombe genes can enhance translation efficiency. Fusion tags affect both expression and purification success—while larger tags (MBP, GST) enhance solubility, they may interfere with folding of small proteins like atp18. Smaller tags (His6, FLAG) minimize structural interference but provide less solubility enhancement.

Proteolytic degradation during purification can be minimized through immediate processing of harvested cells and inclusion of multiple protease inhibitors targeting different classes of proteases. Working at 4°C throughout the procedure reduces proteolytic activity. For purification methodology, researchers should consider that single-step affinity purification rarely achieves sufficient purity; a multi-step approach similar to that used for the Rad18 complex in S. pombe is recommended.

Verification of structural integrity remains crucial, as purified membrane proteins may retain detergent micelles that affect apparent molecular weight on SDS-PAGE. Size exclusion chromatography and dynamic light scattering provide better assessment of protein homogeneity. Finally, activity assays should confirm that purified proteins retain functionality, particularly when reconstitution into liposomes is required for functional studies.

How should researchers interpret complex phenotypes in S. pombe strains with atp18 mutations?

Interpreting complex phenotypes in S. pombe strains with atp18 mutations requires distinguishing primary effects from secondary adaptations while considering cellular context. Researchers should first establish a clear phenotypic baseline through comprehensive characterization of growth, mitochondrial function, and cellular physiology in both fermentable and non-fermentable carbon sources. Temperature sensitivity analysis, similar to studies of rad18 temperature-sensitive mutants , can reveal conditional phenotypes that provide mechanistic insights.

For separating direct from indirect effects, an effective approach utilizes acute versus chronic depletion systems. Comparing phenotypes from an inducible knockout (using systems like the nmt1 promoter) against stable deletion mutants reveals which phenotypes arise immediately from ATP synthase dysfunction versus those developing through compensatory mechanisms. Genetic interaction mapping through synthetic genetic arrays identifies functional relationships; synthetic lethality or suppression with mutations in other genes provides valuable mechanistic insights.

When interpreting bioenergetic measurements, researchers should consider the complete cellular context. Reduced ATP synthesis may trigger multiple adaptive responses, including upregulation of glycolysis, mitochondrial biogenesis, or autophagy. Metabolomic profiling can reveal these adaptations by measuring changes in glycolytic intermediates, TCA cycle metabolites, and ATP/ADP ratios. Transcriptomic analysis identifies genes differentially expressed in response to atp18 mutations, potentially revealing retrograde signaling from mitochondria to nucleus.

For phenotypic rescue experiments, researchers should employ complementation with both wild-type atp18 and site-directed mutants affecting specific functions. Partial rescue phenotypes often indicate that the mutation affects multiple aspects of protein function. Finally, when comparing phenotypes across different genetic backgrounds, remember that laboratory S. pombe strains may contain secondary mutations that influence manifestation of mitochondrial defects.

What statistical approaches are most appropriate for analyzing data from ATP synthase activity assays?

For kinetic analyses measuring parameters like Km, Vmax, or Hill coefficients, nonlinear regression models specific to the reaction mechanism should be employed. When analyzing negative cooperativity in ATP hydrolysis, as observed in S. pombe F1 ATPase , researchers should fit data to appropriate models (e.g., Hill equation with n<1) and compare parameter estimates with their confidence intervals between experimental conditions.

Time-course experiments measuring ATP synthase assembly or activity require repeated measures ANOVA or mixed-effects models that account for both between-group and within-group variations. These approaches are particularly valuable when analyzing assembly kinetics or activity changes under varying conditions over time.

For more complex datasets integrating multiple parameters (e.g., combining ATP synthesis rates, membrane potential, and oxygen consumption), multivariate analysis techniques such as principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) can identify patterns and relationships between variables that might not be apparent in univariate analyses.

Power analysis should be conducted during experimental design to determine appropriate sample sizes. For typical ATP synthase activity measurements, a minimum of biological triplicates with technical duplicates is recommended, though more replicates may be needed depending on the expected effect size and variability. Researchers should report both statistical significance (p-values) and effect sizes (e.g., Cohen's d) to provide complete interpretation of results.

What emerging technologies hold promise for advancing studies of S. pombe ATP synthase structure and function?

Emerging technologies are transforming research on S. pombe ATP synthase structure and function, opening new avenues for understanding this essential mitochondrial complex. Cryo-electron microscopy (cryo-EM) has revolutionized structural biology of macromolecular complexes, enabling visualization of ATP synthase at near-atomic resolution without crystallization. With continued advances in detectors and processing algorithms, cryo-EM will likely soon resolve the structure of S. pombe ATP synthase including small subunits like atp18, revealing their structural contributions to the complex.

Single-molecule techniques offer unprecedented insights into ATP synthase mechanics. High-speed atomic force microscopy can visualize conformational changes during catalysis in real-time, while optical tweezers and magnetic tweezers can measure rotational torque and step size of the rotor elements. Applied to S. pombe ATP synthase, these approaches could reveal species-specific functional adaptations in motor mechanics and efficiency.

Genome engineering advances, particularly CRISPR-Cas9 systems optimized for S. pombe, enable precise modifications from point mutations to domain swaps. Base editing and prime editing technologies allow introduction of specific nucleotide changes without double-strand breaks or donor templates, facilitating detailed structure-function studies of atp18 and other ATP synthase components.

In situ structural techniques like cryo-electron tomography with subtomogram averaging can visualize ATP synthase in its native mitochondrial membrane environment, revealing how supramolecular organization influences function. Advanced fluorescence techniques, including super-resolution microscopy (STORM, PALM) and single-molecule FRET, can track conformational dynamics and subunit interactions in living S. pombe cells.

Mass spectrometry innovations, particularly hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), provide detailed information about protein dynamics, solvent accessibility, and inter-subunit contacts within the ATP synthase complex. Combined with computational modeling approaches like molecular dynamics simulations, these techniques create integrated models of ATP synthase function across different operational states.

How might understanding S. pombe ATP synthase subunit J contribute to therapeutic approaches for human mitochondrial diseases?

Understanding S. pombe ATP synthase subunit J has significant potential to contribute to therapeutic approaches for human mitochondrial diseases through several translational pathways. As a eukaryotic model organism, S. pombe provides a system for studying fundamental aspects of ATP synthase biology that are conserved in humans . The specific advantages of using S. pombe include its genetic tractability, rapid growth, and mitochondrial genome organization that more closely resembles mammals than S. cerevisiae does.

Structure-function studies of atp18 mutants in S. pombe can identify critical residues and domains necessary for ATP synthase assembly and function. These insights allow prediction of pathogenicity for novel variants identified in human ATP synthase genes, helping prioritize candidates for therapeutic development. High-throughput screening platforms using engineered S. pombe strains with humanized ATP synthase components can identify small molecules that stabilize mutant complexes or enhance residual activity.

Understanding ATP synthase supramolecular organization in S. pombe may guide development of therapies targeting oligomerization and membrane organization rather than catalytic activity. Since cristae morphology depends on proper ATP synthase organization, compounds stabilizing these structures could benefit multiple mitochondrial diseases characterized by altered mitochondrial ultrastructure.

S. pombe models of ATP synthase deficiency reveal metabolic adaptations that compensate for reduced oxidative phosphorylation. These compensatory pathways, identified through metabolomic and transcriptomic analyses, represent potential therapeutic targets. For example, if specific metabolic rewiring supports viability in atp18 mutants, similar approaches might benefit patients with ATP synthase disorders.

Gene therapy approaches targeting ATP synthase components can be tested in S. pombe before advancing to mammalian models. The relative simplicity of S. pombe makes it ideal for optimizing delivery methods, expression levels, and functional assessment of gene replacement strategies. Additionally, insights from suppressor screens in S. pombe may identify unexpected genetic modifiers of ATP synthase function that represent novel therapeutic targets for bypassing primary defects in human mitochondrial diseases.