Developing novel methods for systems biology of membrane protein complexes

Mitochondria are the power plants of the cell and play a crucial role in cellular energy conversion and the regulation of apoptosis. 99 % of all mitochondrial proteins are synthesized in the cytosol and have to be imported into mitochondria by a sophisticated import machinery [1,2]. Defects in the protein import are responsible for different mitochondrial diseases and therefore have a direct impact on cell and organism vitality. The protein entry gate of the mitochondria is the translocase of the outer membrane (TOM) complex. Since only little was known about the regulation of TOM-mediated import we conducted a comprehensive phosphoproteome analysis of outer membrane vesicles and isolated TOM complex. Since phosphorylation usually is a substoichiometric process, phosphopeptide enrichment was utilized prior to MS-analysis. As a result, a total of 30 phosphorylation sites could be identified in all components of the TOM complex [3]. Most of the phosphorylation sites could be found on the cytosolic side with casein kinase 2 (CK2) and protein kinase A (PKA) as the primary involved kinases. Therefore, for the first time it could be shown, that mitochondrial protein import is regulated through phosphorylation of TOM components. Based on these findings, quantitative changes of TOM phosphorylation patterns during mitochondrial import are currently analyzed based on label-free and SRM quantification.