Lipid dependent interactions of biomimetic membrane models with the ENTH domain of epsin
von Nelli Teske
Datum der mündl. Prüfung:2019-12-09
Betreuer:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Thomas Burg
EnglischMembrane remodeling processes during endocytosis are highly regulated by the protein-protein and protein-membrane interactions. In clathrin-mediated endocyto-sis (CME) binding of the protein epsin to its receptor lipid phosphatidylinositol-(4,5)-bisphosphate (PIP2) induces the deformation of the membrane. Upon binding of the epsin N-terminal homology domain (ENTH) to PIP2, conformational changes in the protein lead to a newly formed helix, which inserts into the cytosolic leaflet. By using artificial membrane models the binding affinity and the surface topology upon ENTH binding as a function of the lipid composition were analyzed. With in-creasing the PIP2 concentration the experiments on solid supported lipid bi- and monolayers proved a direct relation between the protein occupancy and the lipid content. Subsequently, this indicates that an accumulation of PIP2 on the cytosolic leaflet can facilitate the endocytosis rate. Besides PIP2 also negatively charged lipids with the head group phosphatidylserine (PS) can affect protein binding in endocytosis. In presence of PS, higher binding af-finities and protein occupancies of ENTH to PIP2 doped membranes were observed. Although ENTH is known to act as a monomeric protein, atomic force microscopy (AFM) measurements revealed the appearance of protein clusters induced by PS. Thus, also membrane crowding seems to have an impact on the curvature inducing step in CME. Mutation of the amino acid R114 showed its relevance in ENTH cluster formation as no oligomers were observed with the mutant R114A. Moreover, monolayer penetration experiments were performed to analyze the sur-face activity of ENTH dependent on the lipid composition. Increasing the PIP2 con-tent increased the critical surface pressure. Addition of PS did not significantly in-crease the penetration of ENTH into monolayers in a PIP2 dependent manner, alt-hough a higher protein occupancy on supported lipid bilayers was observed. To investigate whether ENTH has an influence on mechanical properties of mem-branes in the presence of PS, giant unilamellar vesicles (GUVs) were adhered to Neu-trAvidin coated surfaces. Incubation of these GUVs with ENTH resulted in the rup-turing of those due to lipid packing defects in the membrane by helix insertion and PS induced clusters. These results allow to understand how PS alters the binding structure of ENTH to PIP2 doped membranes. This in turn also show that the lipid composition contributes to the regulation of protein-dependent membrane defor-mation during CME.
Keywords: Epsin; ENTH; Lipid dependency; ENTH-membrane interactions