Adaptor-Mediated Control of the Mitochondrial Lon Protease: Coordinating mtDNA Copy Number with Translational Capacity

• Applicant: Professor Dr. Christof Osman
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 570854225

Mitochondrial DNA (mtDNA) encodes essential subunits of the oxidative phosphorylation (OXPHOS) machinery and is therefore critical for mitochondrial energy production. Eukaryotic cells maintain multiple copies of mtDNA; however, the mechanisms that determine and regulate mtDNA copy number (mtDNA CN) remain poorly understood. Using Saccharomyces cerevisiae, we found that deletion of the gene MRX6 leads to increased mtDNA CN. Furthermore, the Mrx6 protein interacts with the mitochondrial Lon protease Pim1 and Mam33, a protein involved in mitoribosome assembly. Structural predictions and biochemical purification suggest that Mrx6 and Mam33 form a subcomplex that associates with the substrate recognition domain of the hexameric Pim1 protease. Importantly, we showed that deletion of MRX6 or MAM33 results in stabilization of the mitochondrial RNA polymerase Rpo41, strongly suggesting that Mrx6 and Mam33 facilitate Pim1-mediated degradation of Rpo41. Based on these results, we aim to pursue two objectives. Our first objective is to characterize the structure and function of the Pim1-Mrx6-Mam33 complex. We will use crosslinking mass spectrometry and cryo-electron microscopy to determine the architecture of the complex and employ in vitro reconstitution assays to assess how Mrx6 and Mam33 influence Pim1 substrate processing. Our second objective is to investigate how the Pim1-Mrx6-Mam33 complex integrates into the broader regulatory network governing mtDNA CN. Since Mrx6, Mam33, and Pim1 have all been linked to mitochondrial translation, we will specifically examine the connection between mtDNA CN regulation and mitoribosome function. We aim to define the role of Mrx6 in mitochondrial translation, test whether defects in translation or ribosome biogenesis trigger changes in mtDNA CN, and analyze how increased mtDNA CN affects mitochondrial gene expression. Specifically, we will test the model that mtDNA CN is determined by translational demands. By focusing on the Pim1-Mrx6-Mam33 axis, this project will provide key mechanistic insight into the regulation of the mitochondrial Lon protease and clarify the interplay between mtDNA CN and mitochondrial translation. These findings will address two fundamental and poorly understood aspects of mitochondrial biology: how mtDNA CN is regulated, and how adapter proteins modulate Lon protease activity.

• DFG Programme: Research Grants