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Forschungsprojekte
A. Funktionelle Regulation von Clathrin-assoziierten Adaptorkomplexen We have recently shown that phosphorylation of AP2 (Fingerhut et al., 2001) by AAK1 (Ricotta et al., 2002) helps to keep the AP complex in a conformation that allows high-affinity binding to Yxx -motif containing cargo membrane proteins when embedded in membranes containing phosphatidyl-inositol-4,5-bisphosphate (PtdIns4,5P2). Binding of AP2 to dileucine signals was also dependent on corecognition with PtdIns4,5P2 but independent of µ2 phosphorylation, strongly arguing that binding occurs at a different site (Höning et al., 2005). Currently, we characterize how the AP2-modulating enzymes are regulated during the formation of a clathrin-coated vesicle.  Figure 1: A model of AP2. Subunits are coloured blue, 2 gold, 2 green, and µ2 purple. The structures of the trunk and the ears were determined separately, the flexible unstructured hinges connecting them are modeled. Indicated are the binding sites of membrane proteins with Yxx -motifs, the phosphorylation on Thr156 by AAK1 in µ2, the putative binding site for membrane protein dileucine motifs, the PIP2 binding site in the very amino-terminal part of -adaptin and the putative phosphorylation sites in the hinges of both large subunits. Accessory and regulatory proteins bind to the ears of both - and ß2 adaptins. Adapted from Owen et al., 2004. B. Analyse von AP4-Funktion in Epithelzellen AP4 is the youngest member of the family of heterotetrameric AP complexes and its function is currently not well understood. We provided evidence for a function is polarized sorting of membrane proteins in epithelial cells (Simmen et al., 2002), other propose a function in glutamate receptor sorting in neurons. To further analyse the function of AP4 we currently apply RNAi and overexpression of tagged-AP4 to investigate its role in epithelial and non-polarized cells. C. Charakterisierung von TGN-Austrittstellen The TGN is a major site for sorting of newly synthesized proteins into different transport routes. Whether heterotetrameric AP complexes act distinct from or in concert with monomeric adaptors to mediate sorting of proteins into different types of TGN-derived vesicles remains to be clarified. In order to address this issue, we currently isolate TGN-derived vesicles in order to characterize their cargo and membrane composition and to trace back their biogenesis. D. Biogenese von Lipidtröpfchen Our recent analysis of mannose-6-phosphate receptor sorting in human cells showed that Tip47, which was previously characterized as a sorting device for the receptors is not relevant at all. Instead we could demonstrate that Tip47 is important for the biogenesis of lipid droplets (Bulankina et al., submitted). Its exact function we try to unravel in this project.
06 September 2011 Stefan Höning Institut für Biochemie I, Joseph-Stelzmann-Strasse 52, D50931 Köln Anregungen und Wünsche: Budi Tunggal  |
