ATAD3 protein family: Molecular dissection of the ATAD3A dual role on the maintenance of mitochondrial ultrastructure and mtDNA-Nucleoid organization

Mitochondria are key organelles in a variety of cellular functions such as ATP production, calcium homeostasis, cell differentiation, and apoptosis. All these functions are possible thanks to the complicated mitochondrial ultrastructure that results in the compartmentalization of the different biochemical processes. An example of this is the cristae compartment that host the oxidative phosphorylation machinery, existing a direct correlation between the cristae density and the respiratory capacity of mitochondria. Moreover, the mitochondrial ultrastructure is crucial for the mitochondrial function, indeed, thinner cristae favor the assembly of respiratory complexes in supercomplexes improving the respiratory efficiency. Therefore, the ultrastructure has become an indicator of the mitochondrial functional status and the study of specific ultrastructural parameters is currently used to describe a dysfunction. In this thesis, by using a dynamic complexomic analysis to identify new cristae remodeling modulators, we have recognized the protein ATAD3A as a highly interesting candidate to participate in the process. Although previous studies pointed to ATAD3A as a regulator of mitochondrial ultrastructure based on results obtained after silencing, no details about the molecular mechanism have been revealed. In addition, ATAD3A have been also implicated in many other cellular processes like cholesterol transport, apoptosis and nucleoid-mtDNA stability. The main goal of this work was to understand whether and how ATAD3A regulates mitochondrial structure and whether this is somehow correlated with its implication in mtDNA stability. To address these questions, we used a variety of biochemical and proteomic approaches, that in combination with proper genetic models have led us to the data presented in this work. We have identified by dynamic complexomic analysis three different complexes of ATAD3A at 1MDa, 500kDa and 250kDa. The observation that only the 500kDa complex is disrupted during the cristae remodeling, and that OPA1 was comigrating in the same disrupted complex, led us to hypothesize its involvement in the cristae maintenance. Indeed, ATAD3A silencing or overexpression was able to decrease or increase the number of cristae, respectively. Moreover, this effect was accompanied by alterations on the nucleoid and mtDNA copy number. By using specific ATAD3A mutant in the coiled-coil (CCD) or ATPase domain of ATAD3A we demonstrated that the ATPase is required for the cristae maintenance while the CCD seems to be involved in nucleoid organization. Our experiments also excluded the participation of the ATAD3A oligomerization in the cristae maintenance. The analysis of the ATAD3A complexes in presence of the different mutants further supported the involvement of the 500kDa complex in the process of cristae maintenance since the ATAD3A ATPase-mutant, that does not maintain cristae does not assemble on it. On the contrary, the CCD-mutant that induces cristae biogenesis assembles in the 500kDa complex. Our data also support the idea that ATAD3A 500kDa complex act as a scaffold protein that allows OPA1 to assemble together cooperating in the cristae biogenesis process. We have also studied the role of the ATAD3A 1MDa complex, which looking in our complexomic analysis, appeared enriched in mitoribosomal and other proteins suggested to be involved in nucleoid and mtDNA stability, suggesting the involvement in the process. To support this idea, cells lacking the mtDNA do not present the ATAD3A 1MDa complex, and also control treated shortly with EtBr display a significant reduction of the same complex. Since the CCD-mutant is not assembling in the 1MDa complex, we analyzed nucleoids in presence of the ATAD3A wild type or mutants after silencing of the endogenous protein. This results further confirm the involvement of the 1MDa in nucleoid organization since the CCD mutant exhibited a decrease in nucleoid are that was not present with the wild type or ATPase-mutant. Overall our data demonstrate a dual role of ATAD3A in cristae biogenesis and nucleoid-mtDNA stability that depends on the different domains: the coiled-coil domain is required for nucleoid organization and the ATPase domain for cristae biogenesis. Additional studies have been done in this PhD thesis to understand whether and how ATAD3B might be able or not to complement the absence of ATAD3A, or if as previously proposed it acts as a dominant negative for ATAD3A. Our work supports the latter hypothesis, that seems to be mediated by the direct interaction of ATAD3A and ATAD3B in the 1MDa complex. Surprisingly, our data show that ATAD3B can revert the pathological mitochondrial phenotype observed in cells expressing the ATPase mutant, suggesting that the nullifying effect of ATAD3B is not specific for the wild type protein. Further experiments are required to confirm these results.