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ESR 2 Project: The role of paraplegin in mitochondrial gene expression.

Work Package 7: Identification of novel factor governing mitochondrial ribosomal assembly and protein synthesis.

Partner: Thomas Langer

Institution: University of Cologne, Germany

Duration: 36 months

Objectives: We want to examine the possibility that mitochondrial genome stability and gene expression depends on a functional domain in the inner membrane, which is formed by prohibitin ring complexes and characterized by a defined protein and lipid composition. Prohibitins ensure the stability of the dynamin-like GTPase OPA1, mitochondrial genome stability and mitochondrial translation, but the molecular basis of these phenotypes is poorly understood. Previous experiments revealed that the function of prohibitins is intimately linked to the metabolism of cardiolipin (CL) and phosphatidylethanolamine (PE). In the present project, we will analyse in depth how prohibitin membrane domains affect mitochondrial genome stability and gene expression. To define how prohibitins affect mitochondrial genome stability translation, we will follow three lines of experiments. First, we will test whether alterations in the lipid composition of the mitochondrial inner membrane destabilize mtDNA and impair translation in the absence of prohibitins. A recently established knockout cell line for a mitochondrial lipid transfer protein regulating PE synthesis in mitochondria revealed an intimate relationship between the accumulation of PE, mitochondrial genome stability and protein synthesis, which will be further investigated. Second, we will examine the role of protein constituents of prohibitin membrane domains for mitochondrial genome stability and translation. We have recently defined the interactome of prohibitins, which includes m-AAA proteases and a number of proteins with unknown functions, which will be characterized functionally. Third, we will employ live cell imaging combined with super-resolution microscopy to define the spatial organization of prohibitin membrane domains, their relative localization to mitochondrial nucleoids as well as their dynamic behaviour.

Expected results: The planned experiments will unravel the role of prohibitin membrane domains and of the lipid composition of mitochondrial membranes for mitochondrial genome stability and gene expression. We will determine how alterations of the general levels of PE and CL and proteins present within prohibitin membrane domains affect the maintenance of mtDNA and mitochondrial translation. These experiments will contribute to unravel general principles of the spatial organization of the mitochondrial inner membrane and their relevance for mitochondrial gene expression. Our project complements efforts of ESR3 who will analyse how m-AAA protease, constituents of prohibitin membrane domains regulate the assembly of mitochondrial ribosomes.