Architecture and function of the mitochondrial VDAC/TSPO super-complex

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The translocator protein 18 kDa (TSPO) is a conserved outer mitochondrial membrane protein which is involved in the regulation of various aspects of mitochondrial and cellular physiology, including bioenergetics, lipid metabolism, cholesterol transport and steroid synthesis, as well as regulation of oxidative stress and Ca2+ homeostasis. Expression levels of the TSPO protein vary in different tissues and during various disease states such as neurodegenerative diseases, inflammatory processes and cancer, as well as neuropsychiatric disorders such as depression. These findings indicate that TSPO provides both general and specific functions related to cellular proliferation, survival and apoptosis, but also influences synaptic transmission and higher brain functions. To be able to fulfill these multifunctional tasks, TSPO has been proposed to be part of a multiprotein super-complex, thereby interacting with protein partners residing in the cytosol, outer mitochondrial membrane (OMM), intermembrane space, inner mitochondrial membrane, and the matrix. One of the best characterized effector proteins interacting with TSPO in the OMM is the voltage-dependent anion channel (VDAC), the major pore in the OMM, constituting the principle gateway for the transport and exchange of ions, ADP/ATP and metabolites. The mechanistic role of TSPO in mitochondrial function and its functional interaction with outer membrane proteins such as VDAC is largely unknown which is also due to missing structural data on the assembly and architecture of the proposed TSPO super-complexes.Due to the need for structural information on TSPO-containing super-complexes in native membrane environment or artificial membrane mimics, and based on our own work on the impact of TSPO ligands on the multiple aspects of mitochondrial and cellular activity, the current project aims at (i) identifying the architecture of the mitochondrial VDAC/TSPO-containing super-complex by means of Cryo-EM single particle analysis, electron crystallography and tomography, (ii) the assembly of the super-complex together with additional proteins, e.g. hexokinase, GSK3beta, ATAD3A, and the ATP/ADP carrier, and (iii) at characterizing the modulatory capacity and the functional interaction within the VDAC/TSPO complex by means of biophysical and pharmacological characterization of VDAC-mediated currents in native and reconstituted protein complexes of defined composition and structure.Our structural and functional studies will shed light on the complex interaction of TSPO and VDAC on a molecular level and will contribute to a deeper understanding of TSPO/VDAC-mediated signaling.