Coordination of protein import and assembly into protein complexes via the mitochondrial presequence import pathway

• Applicant: Professor Dr. Thomas Becker
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 568735694

Mitochondria are essential double membrane-bound cellular organelles that play crucial roles in cellular bioenergetics and are thus key for organismal health. Mitochondria are responsible for the production of the bulk of cellular ATP via oxidative phosphorylation (OXPHOS), a process mediated by the respiratory chain complexes and the F1Fo-ATP synthase. These OXPHOS complexes are major constituents of the mitochondrial inner membrane. The vast majority of their subunits consists of nuclear encoded precursor proteins that typically possess cleavable amino-terminal presequence signals for mitochondrial targeting. These signals drive import into mitochondria through the translocase of the outer membrane (TOM) and the presequence translocase of the inner membrane (TIM23). The presequence translocase-associated motor (PAM) contributes to energize preprotein import into the matrix. After removal of the presequence, the OXPHOS subunits assemble with partner proteins into the mature protein complexes. It is unclear, however, how newly imported OXPHOS subunits proceed to their assembly lines, leading to the formation of mature OXPHOS complexes. The goal of the MitoAssembly project is to address this gap of knowledge providing a systematic description of the coordination mechanisms that ensure dynamic and reliable delivery of newly imported OXPHOS components to their assembly lines. Our groups have recently revealed unexpected roles of subunits of the TIM23 and PAM machineries in this process, pointing to an unprecedented, close link between the TIM23-PAM import route and the assembly of OXPHOS complexes. Here, we will combine the power of genetics, proteomics and biochemical approaches, as well as biophysical probing of imported proteins via in organello EPR spectroscopy, to address the following aims: i) Identification of TIM23/PAM components coordinating import with assembly of OXPHOS subunit. ii) Screening for factors involved in the coupling of import and assembly of OXPHOS subunits. iii) Characterization of the molecular mechanisms coupling the TIM23 and PAM machineries to the OXPHOS assembly lines. iv) Investigation of the regulatory mechanisms that modulate import and assembly of OXPHOS components to meet the cellular bioenergetics demand. Our study will reveal new molecular insights into the biogenesis of OXPHOS complexes, which is highly relevant for the understanding of neurodegenerative diseases, aging and cancers.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partners: Dr. Raffaele Ieva; Dr. Elisabetta Mileo