Mitochondrial inner membrane complex proteomics

Most mitochondrial proteins are synthesized in the cytosol and have to be imported into the organelle by sophisticated mechanisms directing them into the four mitochondrial subcompartments (outer membrane, intermembrane space, inner membrane and matrix). The major preprotein import machinery of the inner mitochondrial membrane, the presequence translocase (TIM23), transports preproteins either across or into the inner membrane. The TIM23 complex switches between different forms depending on the signal information within the imported preprotein. The subunit composition of the different TIM23 forms has been determined earlier by complex purification followed by mass spectrometry and Western Blot analysis. Moreover, the TIM23 machinery was found to be embedded in a membrane protein network that includes respiratory chain supercomplexes and metabolite carriers. Quantitative information on these intricate networks, i.e. the stoichiometry of catalytic subunits and associated partner proteins, is still missing. We aimed to obtain such information by calibrating the analysis of TIM23 isolations with defined amounts of in vitro synthesized proteins known to associate with the TIM23 core complex. For the first time, we will present quantitative data on the composition of the TIM23 machinery and its surrounding membrane protein environment.