InteractiveFly: GeneBrief

OXA1L mitochondrial inner membrane protein: Biological Overview | References

Mitochondrial cristae contain electron transport chain complexes and are distinct from the inner boundary membrane (IBM). While many details regarding the regulation of mitochondrial structure are known, the relationship between cristae structure and function during organelle development is not fully described. This study used serial-section tomography to characterize the formation of lamellar cristae in immature mitochondria during a period of dramatic mitochondrial development that occurs after Drosophila emergence as an adult. The formation of lamellar cristae was associated with the gain of cytochrome c oxidase (COX) function, and the COX subunit, COX4, was localized predominantly to organized lamellar cristae. Interestingly, 3D tomography showed some COX-positive lamellar cristae were not connected to IBM. It is hypothesized that some lamellar cristae may be organized by a vesicle germination process in the matrix, in addition to invagination of IBM. OXA1 protein, which mediates membrane insertion of COX proteins, was also localized to cristae and reticular structures isolated in the matrix in addition to the IBM, suggesting that it may participate in the formation of vesicle germination-derived cristae. Overall, this study elaborates on how cristae morphogenesis and functional maturation are intricately associated. These data support the vesicle germination and membrane invagination models of cristae formation (Jiang, 2020).

Mitochondria are thought to have originated via endosymbiosis. As such, the organelles exhibit unique double-membrane architecture, consisting of outer and inner membranes that are separated by an intermembrane space. The inner membrane can be further subdivided into the inner boundary membrane (IBM) and the cristae invaginations based on ultrastructure, protein composition, and function. In the cristae, electron transport chain (ETC) complexes generate ATP by creating and maintaining a proton gradient between the matrix and the intermembrane space. Importantly, the morphology and remodeling of cristae are indicative of mitochondrial function, and the cristae ultrastructure is heavily influenced by several critical proteins. ATP synthase has been shown to play a structural role in cristae in addition to its enzymatic function, inducing positive membrane curvature at the cristae ridges. Furthermore, the mitochondrial contact site and cristae organizing system (MICOS) complex is known to stabilize the cristae junction, the region where cristae connect to the IBM (Huynen, 2016; Rampelt, 2017; Schorr, 2018). Optic atrophy protein 1 (OPA1), a protein involved in inner membrane fusion, also plays a pivotal role in stabilizing cristae junctions and mediating cristae remodeling during apoptosis. Even though some key proteins have been identified as being essential for the maintenance and remodeling of cristae architecture, the process of functional cristae has not been characterized because the most common model systems do not exhibit distinct stages of cristae formation (Jiang, 2020).

This study examined the process of cristae formation during mitochondrial development in Drosophila after eclosion of adult flies from pupae. At the larval and pupal stages, Drosophila utilizes aerobic glycolysis to support the rapid accumulation of body mass and subsequent metamorphosis. At these stages, the mitochondria in the indirect flight muscle (IFM) scarcely contain lamellar cristae. Beginning at eclosion, mitochondria undergo rapid remodeling in the IFM, establishing densely arranged lamellar cristae that form connective membrane networks. Thus, this physiological time window is highly useful to study the formation of functional cristae in immature mitochondria. Using this model system, this study uncovered several novel aspects of the intricate association between cristae membrane morphogenesis and the acquisition of functionality during mitochondrial development (Jiang, 2020).

This study tracked the dramatic mitochondrial development that occurs in IFM after eclosion of adult Drosophila. Lamellar cristae formation in immature mitochondria was coincidental with the gain of COX activity. This finding was further supported by the observation that the COX4-Apex2 fusion protein was also primarily localized to the organized cristae lamellae of immature mitochondria. It was observed that some COX-positive lamellar cristae were not in contact with the IBM in immature mitochondria. It was therefore hypothesized that some lamellar cristae may form in the matrix by a process other than the invagination of the IBM. In line with this hypothesis, the OXA1 protein, which is known to mediate the membrane insertion of COX subunits, was present in cristae and reticular structures isolated in the matrix additional to the IBM. Overall, this study introduces previously unknown features of the intricately associated processes of cristae membrane morphogenesis and acquisition of functionality during mitochondrial development (Jiang, 2020).

Through evolution, mitochondria have become delicately integrated into eukaryotic cells as organelles with highly functionalized compartments and membranes. Thus, it is no surprise that a sophisticated biomolecular interaction network regulates cristae architecture. One area of active investigation with regard to cristae architecture is the mechanisms of cristae formation. Previously, six theoretical models for the formation and maintenance of cristae were proposed. These models can be grouped into three main categories: 1) the invagination model, where the cristae are formed through the invagination of the IBM; 2) the fusion-mediated model, where the cristae formation is associated with mitochondria fusion; and 3) the vesicle germination model, where vesicles that are either formed de novo in the matrix or by fission from existing cristae fuse with the IBM to establish cristae junctions and cristae. More recently, two other pathways for cristae formation have been proposed based on work in yeast. In one pathway, cristae are formed by the invagination of the IBM, independent of mitochondrial fusion, while in the second pathway, cristae formation involves mitochondrial fusion and OPA1-mediated inner membrane fusion. This work provided an experimental foundation for the first two models previously discussed by Zick, 2009 (Jiang, 2020).

The data in this study do not rule out any of the three categorical models. Furthermore, the data support the existence of vesicle germination-derived cristae, where reticular structures (vesicles) in the matrix may organize into functional lamellar cristae. Therefore, it is hypothesized that cristae are derived from some combination of the invagination and vesicle germination models, in addition to the fusion-mediated model. As such, lamellar cristae may 1) organize at the cristae junction and extend from the IBM as the membranes acquire COX function or 2) extend from the matrix sides by organizing the reticular structures in coordination with OXA1-mediated COX assembly. Lamellar cristae may also 3) form in the matrix as reticular structures, which later contact the IBM and establish MICOS-stabilized cristae junctions (see Hypothetical model of cristae formation). Notably, it is unclear how COX and OXA1 might be transported to the matrix without the involvement of cristae junctions in the vesicle germination model. Thus, the hypothetical model requires further investigation and experimental elucidation (Jiang, 2020).

The generalized mechanism of protein-coupled membrane morphogenesis is well documented in various membrane remodeling processes, such as vesicle budding and fusion. Previously, ATP synthase has been demonstrated to play an essential role in cristae morphogenesis, and cristae morphology reciprocally influences the ETC supercomplex assembly and respiratory efficiency. The intimate connection of cristae morphology with ETC assembly and function is reiterated by observations made in this study. COX complex assembly has been described in great detail and involves the coordination of multiple steps, including protein synthesis, membrane insertion, assembly, and metal incorporation, all of which are mediated by various chaperones and accessory proteins (Soto, 2012). The current study reveals an additional layer of coordination during COX assembly, which is an association with the establishment of cristae ultrastructure. Whether COX assembly directs the lamellar cristae formation or vice versa, or whether the two events are coordinated by a master regulator, remains to be further elucidated (Jiang, 2020).

OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. Insertases constituting a simple cellular system that catalyzes the transmembrane topology of newly synthesized membrane proteins. Depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. This study reports a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. These data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes (Thompson, 2018).

OXA1L is a member of the YidC/Alb3/Oxa1 membrane protein insertase family (Hennon, 2015). The yeast orthologue of OXA1L, Oxa1p, was identified as an important factor for the assembly of complex IV and has also been shown to be important for the assembly of complex V. Oxa1p interacts with the mitoribosome and is required for the co-translational membrane insertion of mitochondrial-encoded Atp6p, Atp9p, Cox1p, Cox2p, Cox3p and Cytb. Oxa1p also appears to have a direct role in the insertion of nuclear-encoded inner mitochondrial membrane (IMM) proteins including Oxa1p itself and an indirect effect on many more IMM proteins including several metabolite transporters, since Oxa1p is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase (Thompson, 2018).

The majority of studies into the function of Oxa1 have been conducted in yeast. Human OXA1L shares 33% identity with the Oxa1p yeast protein and was identified as a homologue by functional complementation and expression of the human gene into an Oxa1p null strain of Saccharomyces cerevisiae. This partially rescued the phenotype of impaired cytochrome c oxidase (COX) assembly, suggesting that OXA1L likely performs a similar role in human cells. Indeed, human OXA1L has since been reported to bind to the mammalian mitoribosome via its C-terminal tail. In contrast to yeast, shRNA-mediated knockdown of OXA1L in human cells was shown to cause a defect in complex I and complex V assembly, but did not affect complex IV. As other insertases may be present in mitochondria, clarification into the importance of OXA1L is required (Thompson, 2018).

This study presents the clinical, biochemical and molecular characterisation of a patient with a severe, childhood-onset mitochondrial encephalopathy and combined respiratory chain deficiency due to biallelic variants in OXA1L identified by WES. Results from cellular and biochemical approaches suggest that OXA1L plays a major role as the insertase for the biogenesis of respiratory chain complexes (Thompson, 2018).

Mutations in OXA1L leads to a severe encephalopathy, hypotonia and developmental delay. The pathogenicity of these variants has been established by showing rescue of the CI, CIV and CV defects in patient fibroblasts after introducing wild-type OXA1L via a retroviral vector (Thompson, 2018).

Interestingly, initial diagnostic tests showed a seemingly isolated complex IV deficiency in patient skeletal muscle, prompting the sequencing of several COX assembly genes. However, subsequent neuropathology experiments indicated an isolated complex I deficiency in the central nervous system. It is also worth noting that while patient skeletal muscle initially appeared to show an isolated complex IV deficiency in terms of activity, Western blotting and BN-PAGE showed a clear complex V defect (which was not assessed by enzymatic assay) and milder defects in complex I in addition to the complex IV defect. The tissue-specific effects of the OXA1L variants remain unexplained, but perhaps suggest that the mitochondrial insertase machinery can vary between tissues, possibly due to differential expression of OXA1L isoforms or that other, as yet unidentified, insertases present in human mitochondria can substitute for OXA1L in different tissues. Interestingly, available mRNA expression data for OXA1L suggest that brain tissue has the lowest relative expression of OXA1L and that the expression pattern is fairly similar between the various isoforms. Recently, more distant homologues of Oxa1 have been identified in the eukaryotic endoplasmic reticulum, namely the DUF106-related proteins WRB/Get1, EMC3 and TMCO1. Similar phylogenetic analysis yielded the same list of homologues with no additional insertase candidates (Thompson, 2018).

The only previous report of OXA1L function in humans was conducted with shRNA depletions in HEK293 cells and demonstrated clear defects in complexes I and V, but complex IV remained unchanged. This was surprising as OXA1L was initially identified as a human gene involved in complex IV assembly through its functional complementation of a complex IV defect in Oxa1p knockout yeast strains. A similar sparing of complex IV was seen in the brain tissue of the patient, with only an isolated complex I defect detected through neuropathology studies, but patient fibroblasts and skeletal muscle showed clear complex IV defects. This study sought to corroborate these findings in patient tissues and to demonstrate the effects of OXA1L depletion on complex IV assembly by other means. Clear defects were seen in complex IV, as well as complexes I and V when OXA1L was depleted using: (i) siRNA in D. melanogaster, (ii) siRNA in human U2OS lines, and (iii) CRISPR/Cas9 in HEK293T cells. Taken together, these data demonstrate that OXA1L is indeed important for complex IV assembly in many human cell types and can act as a general insertase for the majority of mtDNA-encoded subunits into the inner mitochondrial membrane. Similarly, all respiratory complexes including complex IV were affected by depletion of CG6404 (the fly orthologue of OXA1L) in Drosophila. Mitochondrial respiration was strongly affected and flies died shortly (1 or 2 days) after eclosion. These data support an essential role for OXA1L in the assembly of respiratory complexes that have been conserved during evolution (Thompson, 2018).

Immunoprecipitation experiments showed that OXA1L-FLAG interacts with at least nine of 13 mtDNA-encoded proteins along with many nuclear-encoded subunits and assembly factors of complexes I, III, IV and V, suggesting OXA1L may have a role in assembly of these complexes around the co-translational insertion of the core subunits. It was also shown that mitochondrial protein synthesis is not affected in patient fibroblasts, but that the stability of nascent polypeptides is decreased, which is also consistent with a role for OXA1L in co-translational insertion and respiratory complex assembly (Thompson, 2018).

In both siRNA and CRISPR/Cas9 OXA1L knockdown experiments, there was a loss of mitoribosomal proteins, which may be a secondary effect of severe OXA1L depletion since the patient cells with a milder OXA1L depletion do not show loss of MRPs or impaired mitochondrial translation. It is likely that the extreme knockdown of OXA1L is causing loss of MRPs as a downstream secondary effect, possibly due to problems with the mitochondrial membrane potential and/or protein import. This is consistent with the fact that several members of the protein import machinery and certain mitochondrial carriers were also shown to interact with OXA1L in the OXA1L-FLAG immunoprecipitation experiments. Problems with protein import would also account for the decreased SDHA seen in OXA1L-depleted flies. In yeast, Oxa1p has been shown to be required for the biogenesis of the Tim18-Sdh3 module of the TIM22 complex, which is responsible for the import of many inner membrane proteins. This demonstrates that, in yeast, Oxa1p plays a major role in the biogenesis of other inner membrane proteins as a secondary consequence of impaired protein import in Oxa1-deficient mitochondria. The fact that MRP levels are not affected in the patient suggests that the primary effects of OXA1L are in the co-translational insertion of mtDNA-encoded proteins and OXPHOS complex assembly. It is likely that complete loss-of-function mutations are not seen in patients due to more severe mutations not being compatible with life, which is supported by the CRISPR/Cas9 OXA1L knockouts only surviving for 7 days without doxycycline treatment and the death of OXA1L-depleted flies shortly after eclosion (Thompson, 2018).

In yeast, Oxa1p has long been known to be required for the correct insertion of several mtDNA-encoded subunits. Furthermore, Oxa1p plays a crucial role in N-tail protein export in mitochondria, where the N-terminal tail of inner membrane proteins is translocated from the mitochondrial matrix to the intermembrane space. This process applies to nuclear-encoded mitochondrial inner membrane proteins that are imported to the matrix and also to some of the mtDNA-encoded proteins that require the N-terminal tail to reside on the intermembrane space side of the membrane, such as Cox2p. The C-terminal tail of Cox2p is also translocated across the membrane to the intermembrane space, and this process is also impaired in yeast strains lacking Oxa1p. Another member of the Oxa1 insertase family, Cox18p, is also essential for the translocation of the C-terminal tail of Cox2p in yeast. Recently, it has been shown that knockout of COX18 (the human homologue of Cox18p) using TALENs impairs the translocation of the C-terminal tail of COXII in human cell lines (Bourens, 2017). This demonstrates a strong functional conservation in the insertion of COXII between yeast and humans. Knockout of COX18 had no effect on the N-terminal insertion of COXII, suggesting other insertases are involved in this process. Since Oxa1p is essential for N-terminal insertion of Cox2p in yeast and the homologues Cox18p and COX18 are functional orthologs, it is likely that OXA1L is at least partially responsible for N-terminal insertion of COXII in humans. These data are consistent with this notion as COXII is decreased in patient fibroblasts, skeletal muscle and OXA1L siRNA-depleted U2OS cells, and COXII was significantly enriched in the OXA1L-FLAG immunoprecipitation experiments. OXA1L is not only important for COXII insertion, but is more likely to be a more generalised insertase similarly to Oxa1p in yeast (Thompson, 2018).

Search PubMed for articles about Drosophila OXA1L

Hennon, S. W., Soman, R., Zhu, L. and Dalbey, R. E. (2015). YidC/Alb3/Oxa1 Family of Insertases. J Biol Chem 290(24): 14866-14874. PubMed ID: 25947384

Huynen, M. A., Muhlmeister, M., Gotthardt, K., Guerrero-Castillo, S. and Brandt, U. (2016). Evolution and structural organization of the mitochondrial contact site (MICOS) complex and the mitochondrial intermembrane space bridging (MIB) complex. Biochim Biophys Acta 1863(1): 91-101. PubMed ID: 26477565

Jiang, Y. F., Lin, H. L., Wang, L. J., Hsu, T. and Fu, C. Y. (2020). Coordinated organization of mitochondrial lamellar cristae and gain of COX function during mitochondrial maturation in Drosophila. Mol Biol Cell 31(1): 18-26. PubMed ID: 31746672

Rampelt, H. and van der Laan, M. (2017). The Yin & Yang of Mitochondrial Architecture - Interplay of MICOS and F1Fo-ATP synthase in cristae formation. Microb Cell 4(8): 236-239. PubMed ID: 28845421

Schorr, S. and van der Laan, M. (2018). Integrative functions of the mitochondrial contact site and cristae organizing system. Semin Cell Dev Biol 76: 191-200. PubMed ID: 28923515

Soto, I. C., Fontanesi, F., Liu, J. and Barrientos, A. (2012). Biogenesis and assembly of eukaryotic cytochrome c oxidase catalytic core. Biochim Biophys Acta 1817(6): 883-897. PubMed ID: 21958598

Thompson, K., Mai, N., Olahova, M., Scialo, F., Formosa, L. E., Stroud, D. A., Garrett, M., Lax, N. Z., Robertson, F. M., Jou, C., Nascimento, A., Ortez, C., Jimenez-Mallebrera, C., Hardy, S. A., He, L., Brown, G. K., Marttinen, P., McFarland, R., Sanz, A., Battersby, B. J., Bonnen, P. E., Ryan, M. T., Chrzanowska-Lightowlers, Z. M., Lightowlers, R. N. and Taylor, R. W. (2018). OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect. EMBO Mol Med 10(11). PubMed ID: 30201738

Zick, M., Rabl, R. and Reichert, A. S. (2009). Cristae formation-linking ultrastructure and function of mitochondria. Biochim Biophys Acta 1793(1): 5-19. PubMed ID: 18620004

date revised: 12 March 2020

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