Universität zu Köln 

Prof. Dr. Stefan Höning

Institut für Biochemie I, Universitätsklinikum Köln
email: shoening@uni-koeln.de
phone: +49-(0)221 478 3656
website

In this project, we focus on the regulatory mechanisms that modulate the functions of clathrin adaptors, which are key factors in the formation of clathrin-coated vesicles formed at the plasma membrane, at endosomes and the trans side of the Golgi. Among the 40 clathrin adaptors that are expressed in mammalian cells, the family of the monomeric GGA proteins (GGA1-3) and the family of the heterotetrameric adaptor complexes (AP1-4) share the ability to recruit cargo membrane proteins and numerous other factors, including the coat protein clathrin, to the sites of vesicle formation. In this context, the recognition of a specific membrane, the binding to cargo as well as the interaction with accessory vesicle-associated proteins and clathrin needs to be tightly controlled both spatially and temporally. We started to address these issues using AP2 as a prototype. AP2 is restricted to the plasma membrane and is a key player in the formation of endocytic clathrin-coated vesicles. We combined structural analyses with functional in vitro assays to show that AP2 exists in two conformations, a closed form present in the cytosol and an open – membrane-associated form. It requires a critical concentration of phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2) in the membrane to initiate membrane recruitment of AP2, which is mediated by two PtdIns4,5P2 binding sites in the complex. Subsequently, the whole AP2 complex undergoes a large conformational change: it collapses inwards and the c-terminal fragment of the µ2-subunit is completely relocalised. As a result, AP2 exposes a surface with four binding sites for PtdIns4,5P2 and the binding sites for tyrosine- and acidic dileucine sorting signals, all becoming co-planar orientated towards the membrane. Kinetic analyses suggest that 99.9% of cytosolic AP2 is in the closed conformation, in which the two sorting signal binding sites are blocked by parts of the ß2 subunit. This provides an easy mechanism to avoid non-specific binding of AP2 to cytosolic proteins that expose sorting signal like sequences on their surface.

Our results on AP2 suggest that PtdIns4,5P2 is the major determinant for membrane recruitment. However, another factor that is discussed in this scenario is the small GTPase Arf6. We therefore like to analyse whether indeed Arf6 functions in recruitment of AP2 to the plasma membrane. Another prominent member of the Arf family of GTPases is Arf1, which is believed to function as a recruitment factor of the other intracellular APs (AP1, AP3, AP4) and the monomeric GGAs. However, we assume that neither Arf1 nor a given phosphoinositide species is sufficient to determine the specific localisation of the intracellular GGAs and APs. We will therefore use cell-based and in vitro assays involving liposomes to address the significance of Arf1, of individual phosphoinositide species and other potentially yet unknown recruitment factors. Most of the known AP-interacting proteins bind to short sequences in the appendage domains of the large subunits, but it is unclear whether these parts are always available. We like to address the possibility of appendage binding to the core part of the AP complexes, which would provide an easy mechanism in the control of effector binding.

Running time: 01/2006 – 06/2015

Recent publications:

Hackmann, Y., Graham, S.C.,  Ehl, S., Höning, S., Lehmberg, K., Arico, M., Arico, Owen, D.J.,  Griffiths, G.M. (2013). Syntaxin binding mechanism and disease-causing mutations in Munc18-2. PNAS 110, 4482-91. PubMed

Miller, S.E., Sahlender, B. D.A., Graham, S.C., Höning, S., Robinson, M. S., Peden, A.A. and Owen., D. (2011). The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM. Cell 147, 1118-113.

Jackson, L.P., Kelly, B.T., McCoy, A.J., Gaffry, T., James, L.C., Collins, B.M., Höning, S., Evans, P.R., and Owen, D.J. (2010). A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex. Cell 141, 1220-1229.

Bulankina, A.V., Deggerich, A., Wenzel, D., Mutenda, K., Wittmann, J.G., Rudolph, M.G., Burger, K.N., and Höning, S. (2009). TIP47 functions in the biogenesis of lipid droplets. J. Cell Biol. 185, 641-655.

Katoh, Y., Ritter, B., Gaffry, T., Blondeau, F., Höning, S., and McPherson, P.S. (2009). The clavesin family, neuron-specific lipid- and clathrin-binding Sec14 proteins regulating lysosomal morphology. J. Biol. Chem. 284, 27646-27654.

Edeling, M.A., Sanker, S., Shima, T., Umasankar, P.K., Höning, S., Kim, H.Y., Davidson, L.A., Watkins, S.C., Tsang, M., Owen, D.J. et al. (2009). Structural requirements for PACSIN/Syndapin operation during zebrafish embryonic notochord development. PLoS One 4, e8150.

Kelly, B.T., McCoy, A.J., Spate, K., Miller, S.E., Evans, P.R., Höning, S., and Owen, D.J. (2008). A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex. Nature 456, 976-979.

Chapuy, B., Tikkanen, R., Mühlhausen, C., Wenzel, D., von Figura, K., and Höning, S. (2008). AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways. Traffic 9, 1157-1172.

Ricotta, D., Hansen, J., Preiss, C., Teichert, D., and Höning, S. (2008). Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2. J. Biol. Chem. 283, 5510-5517.

Icking, A., Amaddii, M., Ruonala, M., Höning, S., and Tikkanen, R. (2007). Polarized transport of Alzheimer amyloid precursor protein is mediated by adaptor protein complex AP1-1B. Traffic 8, 285-296.