P03 - Heterogeneity in protonic energy coupling

The aim of this project is to better understand the origin and mode of operation of the proton motor force, PMF. This topic is important in the context to understand the heterogeneity of mitochondria in general, the question of local quality control and the elimination of dysfunctional mitochondrial sections and the importance of individual cristae as microcompartments. We would like to examine the following questions: Is the lateral PMF along the inner membrane the same at all sites (i), is the transmembrane PMF the same at all sites (ii), is the PMF under steady state respiratory conditions different at different proton pumps (iii), and what influence does the ultrastructure of the inner membrane and the microcompartmentation of protein complexes have on the local proton concentration (iv)? To investigate this, we will create a detailed pH map for the different structural and functional regions of the mitochondrial inner membrane using highly sensitive, genetically encoded pH sensors that are already successfully used in our laboratory. Of particular interest is whether the peripheral intermembrane spaces, cristae junctions and cristae domains with pronounced membrane curvature display different PMF. To determine pH values, the pH-sensitive GFP derivative superecliptic pHluorin is fused with proteins that can be assigned to specific compartments. For simultaneous pH determination on the matrix side, we will generate specific HaloTag fusion proteins which will be stained posttranslationally with pH-sensitive SNARF-1HTL. Compartment-specific candidates for local pH measurement include subunits of the inner membrane protein import machinery, TIM, the MICOS complex at cristae-junctions and the OXPHOS complexes (cristae lumen and matrix space). The obtained pH profiles of the inner membrane will be correlated with the localization profile of OXPHOS complexes obtained by single molecule tracking and localization microscopy. An important aspect will be to understand how changes in membrane architecture affect the pH profiles of different inner membrane regions and ultimately the bioenergetic function.

We are convinced that the group of specialists in this research network will not only support this project from different sides, but that their expertise is essential for progress: In order to decipher the effects of a defective OXPHOS organization on PMF and functionality, we will work closely with P. Rehling and S. Jakobs. Bioenergetic consequences of cristae-junction changes in MICOS mutants are analysed together with M. Meinecke. In cooperation with K. Winklhofer we will investigate OXPHOS/PMF changes in NFBkappa-b-mediated stress reactions. Together with O. Daumke we will investigate the importance of OPA1 for the maintenance of PMF. The aim is to answer whether and how the pro-apoptotic role of OPA1 is related to changes in PMF.