Proteomic characterization of mitochondrial ion channels

Mitochondria are key organelles with elementary roles in the regulation of pivotal cellular processes like nutrition metabolism, energy production, signal transduction, ion regulation and programmed cell death. This outstanding capacity is primarily depending on the function of mitochondrial membrane proteins, at which the mitochondrial transporters encoded by the SLC25 genes play fundamental parts. A common feature of the SLC25 family proteins is to provide a link between mitochondria and the cytosol by facilitating the flux of a large variety of solutes, ranging from H+ to ATP, through the permeability barrier of the inner mitochondrial membrane. Fascinatingly, for some of the SLC25 family members, the phosphate carrier, the adenine nucleotide translocators, and the uncoupling proteins 2 and 3, evidence exist showing a participation of these proteins in the function of mitochondrial ion channels, by being components of heteromultimeric protein complexes. All of these proteins are linked to mitochondrial ion channels involved in the regulation of Ca2+ signal transduction pathways, the former two as constituents of the mitochondrial permeability transition pore (mPTP), which is involved in the Ca2+-dependent execution of cell death, and the latter have been shown to be fundamental for the main mitochondrial Ca2+ uptake route, the mitochondrial Ca2+ uniporter (MCU). Despite of these findings, our knowledge about the channels` entire constitution is limited and still needs to be addressed. Accordingly, work package A of this proposal, which will be performed in the laboratory of Prof. Andrew Emili, at the University of Toronto, Canada, is designed to investigate the molecular composition of both the MCU and the mPTP by utilizing a proteomics approach, comprising the tandem affinity purification of the tagged SLC25 proteins mentioned above, and a subsequent analysis of the proteins as well as intimately associated proteins by mass spectrometry methodologies. Additionally, tandem mass spectrometry-based sequencing of protein samples will provide important information on defined post-translational modifications of the channels` constituents and thus would give further insight into the regulation of these signaling complexes. Work package B, proposed for the return phase of the fellowship, is designed to assess the contribution of the individual protein complex subunits, derived from work package A, to the function of the respective mitochondrial channel. The projects outcome will reveal insights into the protein composition and the principles of the regulation of the MCU and the mPTP. The molecular characterization will foster our understanding on the physiological function of these channels and provides indispensable information on the mechanisms that are responsible for the channels` dysfunction, which may affect or accelerate the development and progression of certain diseases, like cardiovascular diseases, neuro-degenerative diseases and cancer.