Abstract
Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P(2) specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H(0)) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P(2)-dependent membrane penetration of H(0) is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P(2)-independent manner. Depletion of PtdIns(4,5)P(2) from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P(2) concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.
Highlights
The roles of anionic lipids in the actions of N-BAR domains are not fully understood
After Giant Unilamellar Vesicle (GUV) were sedimented in the bottom of the well, proteins were added gently, and the entire well was scanned with an automated x-y stage (2-min scan time) at 37 °C, and images were captured every 5 s with a charge-coupled device camera controlled with Metamorph software (Roper Scientific)
We quantitatively determined the PtdInsP selectivity in terms of Kd by equilibrium Surface Plasmon Resonance (SPR) analysis. Both dAmp-BAR and EndoA1-BAR have the highest affinity for vesicles containing PtdIns(4,5)P2 and PtdIns(3,4,5)P2: i.e. 3 mol % of these lipids caused a Ϸ3-fold increase in their vesicle affinity over POPC/POPS (8:2) vesicles
Summary
The roles of anionic lipids in the actions of N-BAR domains are not fully understood. Amphiphysins (15) and endophilins (22–24) have 35– 40 N-terminal residues that are important for their potent vesicle tubulation activity (Fig. 1, A and B) These residues were not defined in the crystal structures of dAmp-BAR and endophilin A1-BAR (EndoA1-BAR), they are predicted to form an amphiphilic ␣-helix (H0) (see Fig. 1, C and D) and penetrate the membrane (15, 23, 24) as is the case with H0 of the ENTH domain of epsin[1] (14). Despite remarkable success in structural characterization of various BAR domains, questions still remain as to how exactly these domains interact with cell membranes containing various lipids and whether they induce membrane deformation by a universal mechanism or by different mechanisms To address these mechanistic questions, we investigated two structurally well characterized N-BAR domains, dAmp-BAR and EndoA1-BAR, with major lipid components of the plasma membrane, including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylserine (PS). Our biophysical and quantitative fluorescence imaging studies of the two N-BAR domains and respective mutants provide new mechanical insight into the regulatory role of PtdIns(4,5)P2 in their membrane interaction and deformation and the differential mechanisms by which the two N-BAR domains cause membrane binding and deformation
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