Structural and functional studies of import and intermembrane space transfer of mitochondrial membrane proteins

• Applicant: Professor Dr. Nils Wiedemann
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2018 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 406757425

Mitochondria are essential for Fe-S cluster biogenesis, crucial for apoptosis, involved in numerous metabolic pathways and well known for their role in ATP synthesis. All these processes depend on beta-barrel channels in the outer membrane and alpha-helical metabolite carriers in the inner membrane. Similar to the vast majority of mitochondrial proteins, all these channels and transporters are encoded in the nucleus and translated on cytosolic ribosomes. Classical mitochondrial precursor proteins contain an N-terminal presequence which is required and sufficient for targeting and import and afterwards it is cleaved off to produce the mature protein. The goal of this project is a comprehensive description of the import of mitochondrial membrane protein precursors with internal targeting signals. After targeting to the translocase of the outer membrane (TOM) and translocation these hydrophobic precursors are sorted through the aqueous intermembrane space. Subsequently, beta-barrel precursors are inserted with the help of the sorting and assembly machinery (SAM) into the outer and alpha-helical metabolite carriers by the carrier translocase (TIM22) into the inner membrane. Major open questions are the specific targeting mechanisms of the three Tom receptors for membrane proteins with internal targeting signals, the structural basis of protein chaperoning in the intermembrane space by the TIM chaperone system and the transfer to the membrane insertase complexes.By an integrated structural biology and biochemical approach, we will analyze the contribution of the different redundant Tom receptors for the targeting of beta-barrel proteins and metabolite carriers. Moreover, we will determine the mechanism of membrane protein precursor transfer by the TIM chaperone system from the TOM complex through the aqueous intermembrane space to the SAM and TIM22 complexes. We will analyze the structure and dynamics of the complexes formed by the membrane protein precursors and the receptor domains and chaperones by combining NMR, SAXS and other biophysical approaches, determine relative binding affinities, and study the preprotein transfer from chaperones to the Sam50-POTRA domain and to the Tim54 receptor of the TIM22 complex at the structural level. These biophysical and structural approaches will be complemented by generation of site-specific point mutants of the receptor and chaperone proteins and their in vivo and in vitro analysis by growth assays and import experiments into isolated mitochondria. Taken together, we will complement the knowledge for the import of the classical mitochondrial precursor proteins by an in-depth description of the import of the two most abundant membrane protein classes of mitochondria and this will reveal important principles also for chloroplasts and Gram negative bacteria.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partners: Dr. Beate Bersch; Professor Paul Schanda, Ph.D.