Recombinant Mouse Mitochondrial inner membrane protein OXA1L (Oxa1l)

What is OXA1L and what is its primary function in mitochondria?

OXA1L (Mitochondrial inner membrane protein OXA1L) is the mammalian homolog of the yeast Oxa1 protein and a member of the YidC/Alb3/Oxa1 membrane protein insertase family. It serves as a critical insertase in the mitochondrial inner membrane, facilitating the cotranslational insertion of mitochondrial-encoded polypeptides into the inner membrane . OXA1L spans the inner membrane five times, with its C-terminal domain exposed to the mitochondrial matrix, providing a platform for binding to mitochondrial ribosomes . Through this interaction, OXA1L facilitates the insertion of mitochondrial translation products into the lipid phase, playing an essential role in the biogenesis of the oxidative phosphorylation (OXPHOS) machinery .

Which respiratory chain complexes require OXA1L for proper assembly?

Recent research demonstrates that OXA1L is required for the assembly of multiple respiratory chain complexes. While earlier studies in yeast suggested OXA1 was primarily required for complexes IV and V assembly, and initial human studies indicated roles in complexes I and V, more comprehensive analyses reveal broader involvement . Patient studies and targeted depletion experiments in human cells and Drosophila melanogaster show that OXA1L plays critical roles in the assembly of complexes I, IV, and V of the respiratory chain . Immunoprecipitation experiments with OXA1L-FLAG have demonstrated interactions with at least nine of 13 mtDNA-encoded proteins along with many nuclear-encoded subunits, confirming its broad involvement in respiratory complex assembly .

How does OXA1L interact with the mitochondrial ribosome?

OXA1L interacts with the mitochondrial ribosome through its C-terminal domain, which extends into the mitochondrial matrix. Structural analyses of the mitochondrial ribosome have provided molecular information on this association, suggesting that OXA1L binds to the mitochondrial large subunit (mtLSU) through interactions with proteins mL45 and uL23m . This interaction is translation-independent, as demonstrated by experiments with thiamphenicol (TAP), which inhibits mitochondrial translation. When mitochondrial translation is inhibited, OXA1L still copurifies with mitochondrial ribosomes, indicating that the association is maintained independent of translation activity .

What is the relationship between OXA1L and TMEM126A, and how does this impact mitochondrial protein biogenesis?

TMEM126A has been identified as a novel OXA1L-interacting protein that forms a complex with the OXA1L insertase in mitochondria . This interaction is independent of active mitochondrial translation or OXA1L association with the ribosome, as demonstrated by experiments using translation inhibitors and ribosome-depleted cells . TMEM126A associates with mitochondrial ribosomes and translation products, and its loss leads to destabilization of mitochondrial translation products, triggering an inner membrane quality control process . The OXA1L-TMEM126A complex appears to be crucial for proper insertion of mitochondrial-encoded proteins into the inner membrane, as depletion of TMEM126A affects the stability of newly synthesized mitochondrial proteins and impacts respiratory chain complex assembly .

How do mutations in OXA1L affect mitochondrial function and what are the clinical manifestations?

Pathogenic mutations in OXA1L have been associated with severe mitochondrial encephalopathy, hypotonia, and developmental delay . A patient harboring biallelic variants in OXA1L (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) presented with severe encephalopathy and died at age 5, showing complex IV deficiency in skeletal muscle . At the molecular level, these mutations lead to decreased OXA1L protein levels and reduced steady-state levels of subunits of complexes IV and V .

Interestingly, tissue-specific effects of OXA1L variants have been observed, with skeletal muscle showing primarily complex IV deficiency while brain tissue displayed isolated complex I deficiency . This tissue specificity may result from differential expression of OXA1L isoforms or the presence of other unidentified insertases that can partially compensate for OXA1L deficiency in different tissues .

What mechanisms underlie cristae formation in relation to OXA1L function?

OXA1L plays a role in cristae morphogenesis and functional maturation of mitochondria. Evidence from Drosophila studies suggests that the formation of lamellar cristae is associated with the gain of cytochrome c oxidase (COX) function, with the COX subunit COX4 predominantly localized to organized lamellar cristae . 3D tomography analyses have revealed that some COX-positive lamellar cristae are not connected to the inner boundary membrane (IBM), suggesting that lamellar cristae may form not only through invagination of the IBM but also potentially through a vesicle germination process in the matrix .

OXA1L, which mediates membrane insertion of COX proteins, has been localized to cristae and reticular structures isolated in the matrix in addition to the IBM, suggesting its participation in the formation of vesicle germination-derived cristae . These findings indicate that OXA1L contributes to both cristae structure and function during mitochondrial development, supporting both vesicle germination and membrane invagination models of cristae formation .

What are effective strategies for tagging OXA1L without disrupting its function?

When designing tagged versions of OXA1L for experimental studies, the position of the tag is critical to maintain protein functionality. Research has shown that N-terminally tagged versions of OXA1L (e.g., FLAGOXA1L with the tag introduced adjacent to the predicted presequence in the N-terminal region at amino acid 74) efficiently purify the mitochondrial ribosome and membrane integral early assembly factors like MITRAC12 and C12ORF73 .

In contrast, C-terminally tagged OXA1L versions are less efficiently purified and show reduced interaction with ribosomal proteins . This is consistent with the understanding that the C-terminus of OXA1L mediates interaction with the mitochondrial ribosome, and tagging this region can interfere with ribosome association . For optimal results in immunoprecipitation and interaction studies, N-terminal tagging is recommended to preserve OXA1L's functional interactions with ribosomes and other mitochondrial proteins.

How can researchers effectively assess OXA1L's role in mitochondrial protein insertion?

To evaluate OXA1L's role in mitochondrial protein insertion, researchers can employ several complementary approaches:

• siRNA-mediated depletion: Using targeted siRNA against OXA1L in cell lines such as U2OS cells effectively depletes OXA1L and allows for assessment of the impact on respiratory chain complex assembly and mitochondrial translation products .

• Immunoprecipitation with tagged OXA1L: Using N-terminally tagged OXA1L (FLAGOXA1L) enables efficient purification of the protein along with associated ribosomes and early assembly factors, allowing for the identification of interaction partners through mass spectrometry or western blot analysis .

• Rescue experiments: Expressing wild-type OXA1L in patient fibroblasts with OXA1L mutations can demonstrate causality and confirm pathogenicity of variants. This approach has successfully shown the rescue of complex IV and V defects in patient fibroblasts transduced with wild-type OXA1L .

• Model organisms: Using model organisms like Drosophila melanogaster for targeted depletion of OXA1L can provide insights into the in vivo effects on respiratory chain complex assembly and mitochondrial function .

• SILAC-based quantitative proteomics: Stable isotope labeling with amino acids in cell culture (SILAC) combined with mass spectrometric analysis of OXA1L immunoprecipitates can define the OXA1L interactome in a quantitative manner .

What techniques are most effective for analyzing OXA1L-dependent respiratory chain complex assembly?

Multiple complementary techniques can be used to comprehensively analyze OXA1L-dependent respiratory chain complex assembly:

• Blue Native PAGE (BN-PAGE): This technique allows visualization of intact respiratory chain complexes and can reveal defects in complex assembly in OXA1L-deficient cells or tissues .

• Western blotting: Assessing steady-state levels of subunits of respiratory chain complexes can indicate assembly defects. This has revealed that OXA1L depletion affects levels of complex I (NDUFB8, NDUFA5, NDUFB10), complex IV (COXII), and complex V subunits .

• Enzymatic activity assays: Measuring the activities of respiratory chain complexes provides functional assessment of assembly defects. Complex IV activity assays in particular have been used to demonstrate OXA1L's importance for cytochrome c oxidase function .

• Immunoprecipitation of OXA1L: This technique reveals the enrichment of mtDNA-encoded subunits of complexes I, IV, and V, demonstrating OXA1L's direct role in inserting these components into the respective complexes .

• Serial-section tomography: This advanced imaging technique can characterize the formation of lamellar cristae in mitochondria and correlate cristae structure with respiratory chain complex function, particularly for studying COX localization in relation to OXA1L function .

How can researchers distinguish between primary OXA1L defects and secondary OXPHOS deficiencies?

Distinguishing primary OXA1L defects from secondary OXPHOS deficiencies requires a multifaceted approach:

• Complementation studies: Expressing wild-type OXA1L in patient-derived cells can rescue OXPHOS defects in primary OXA1L deficiencies but not in secondary deficiencies. This approach has been used to confirm the pathogenicity of OXA1L variants by showing rescue of complex IV and V defects in patient fibroblasts transduced with wild-type OXA1L .

• Pattern of respiratory chain defects: Primary OXA1L defects typically affect multiple respiratory chain complexes (I, IV, and V), though the pattern may vary between tissues. In contrast, secondary OXPHOS deficiencies may show different patterns depending on the primary cause .

• Tissue-specific analyses: Comprehensive assessment across multiple tissues can help identify the characteristic tissue-specific pattern of respiratory chain defects seen in OXA1L mutations (e.g., complex IV deficiency in skeletal muscle but complex I deficiency in brain tissue) .

• Molecular analysis of OXA1L: Sequencing the OXA1L gene and quantifying OXA1L protein levels can directly identify pathogenic variants and protein deficiency, distinguishing it from secondary causes .

• Analysis of mitoribosomal proteins: OXA1L depletion affects levels of mitoribosomal proteins (MRPs), which can serve as an additional marker to distinguish from other OXPHOS deficiencies .

What is the relationship between OXA1L dysfunction and mitochondrial quality control pathways?

OXA1L dysfunction triggers specific mitochondrial quality control pathways to deal with improperly inserted mitochondrial proteins:

• Inner membrane protein quality control: Loss of TMEM126A, an OXA1L-interacting protein, leads to destabilization of mitochondrial translation products and triggers an inner membrane quality control process, in which newly synthesized proteins are degraded along with the OXA1L insertase .

• OXA1L turnover: In the absence of proper insertion machinery components like TMEM126A, the OXA1L protein itself is turned over, suggesting a feedback mechanism to prevent accumulation of dysfunctional insertase .

• Tissue-specific compensation: The observation of tissue-specific effects of OXA1L variants suggests the existence of compensatory mechanisms that may include differential expression of OXA1L isoforms or alternative insertases that can substitute for OXA1L in different tissues .

• Cristae remodeling: OXA1L contributes to cristae morphogenesis, and dysfunction may impact cristae structure and organization. This structural alteration could trigger quality control pathways that monitor mitochondrial membrane integrity .

What are the most promising approaches for therapeutic intervention in OXA1L-related disorders?

While current research does not directly address therapeutic interventions for OXA1L-related disorders, several approaches might be considered based on our understanding of OXA1L function:

How might novel imaging techniques advance our understanding of OXA1L's role in cristae formation?

Advanced imaging techniques offer promising avenues to further investigate OXA1L's role in cristae formation:

• Super-resolution microscopy: Techniques like STED (Stimulated Emission Depletion) or PALM (Photoactivated Localization Microscopy) could provide nanoscale resolution of OXA1L localization relative to cristae membranes and other mitochondrial structures.

• Cryo-electron tomography: This technique could provide high-resolution 3D visualization of OXA1L's interaction with ribosomes and insertion of proteins into the inner membrane, potentially capturing the process of cristae formation in relation to protein insertion.

• Live-cell imaging with tagged OXA1L: Using minimally disruptive tags and advanced live-cell imaging could reveal dynamic aspects of OXA1L function during mitochondrial development and cristae formation.

• Correlative light and electron microscopy (CLEM): This approach could connect the molecular specificity of fluorescence microscopy with the structural context provided by electron microscopy, offering insights into how OXA1L participates in both vesicle germination and membrane invagination models of cristae formation .