Towards elucidation of the biogenesis pathway of human mitochondrial ATP synthase

• Applicant: Dr. Dejana Mokranjac
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 568963472

ATP, the universal energy currency of living organisms, is mainly produced by the ATP synthase. In eukaryotic cells, this evolutionary conserved molecular machine is present in the mitochondrial inner membrane. In humans, it consists of 18 different proteins that are encoded either in the mitochondrial or in the nuclear genome and assemble to form a 28-subunits complex. Subunit c (Su.c) is the central component of the complex which forms the membrane-embedded ring consisting of 8 protein copies (c8-ring). Not surprisingly, not only mutations that directly affect the function of ATP synthase but also its assembly have direct consequences on human health. Yet, the assembly process of the ATP synthase is still poorly understood. Mutations in TMEM70, causing neonatal mitochondrial encephalo-cardiomyopathies, are the most frequent cause of ATP synthase deficiencies of nuclear origin. We have recently uncovered that TMEM70 plays a crucial role in the biogenesis of the c8-ring. However, at which step(s) of Su.c biogenesis, from its import from the cytosol, over insertion into the inner membrane and oligomerization into a c8-ring, to the assembly of the ring into the final ATP synthase, TMEM70 is involved in has remained unknown. In addition, a recent report and our preliminary data suggest that two additional factors may influence biogenesis of Su.c. Here, we will combine our expertise to elucidate the biogenesis pathway of human ATP synthase, with a specific focus on Su.c and its three recently identified biogenesis/assembly factors. We will focus on four main aims: 1. Using a battery of established assays that evaluate the cellular and mitochondrial bioenergetics, we will elucidate the consequences patient-derived TMEM70 mutations have on cellular and mitochondrial levels. 2. Using microscopy techniques, we will determine the sub-mitochondrial localizations of the recently identified biogenesis/assembly factors of Su.c, investigate changes in their localization under various conditions, and explore their potential colocalization and overlapping functions at the cellular level. 3. Using our recently developed import assay of in vitro synthesized radiolabeled precursor of Su.c into isolated human mitochondria, we will elucidate the steps and kinetics of Su.c import, membrane insertion, oligomerization and assembly into the ATP synthase, and identify the roles the recently identified biogenesis/assembly factors play in this process. 4. Using cell-free expression of Su.c and TMEM70 and their mutants in the presence of nanodiscs, we will elucidate the roles of individual domains of TMEM70 in the c8-ring biogenesis and characterize the supramolecular assemblies incorporated into nanodiscs using electron microscopy. This unique combination of approaches will enable the dissection of the biogenesis pathway of ATP synthase, clarifying one of the most basic cellular processes and potentially offering insights into associated pathogenic mechanisms.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partners: Dr. Stephane Duvezin-Caubet; Dr. Marie-France Giraud