Investigating How Membrane Localization Regulates Protein Assembly During Clathrin-Mediated Endocytosis

Abstract

Cell growth, development and proper cellular function depends heavily on the precisely controlled mechanisms of entry and exit of macromolecules into and out of cells. Clathrin mediated endocytosis (CME) is one such process that mediates the entry of many biologically important molecules, including growth factors, nutrient molecules and cell signaling molecules into cells. Although CME is a well-studied, multi-step process, it is still not known how the localization of components to the membrane surface can modify the strength of protein interactions and control the speed and stability of clathrin-coat assembly. It has been observed that when proteins are on the membrane surface, binding rate constants and equilibrium constants (Ka2D) are fundamentally distinct from their solution values (Ka3D). The Ka2D values control association strength between proteins on the surface and yet they have rarely been measured experimentally for any proteins. Our group has developed a theory to obtain the Ka2D from the experimentally obtained binding strengths (Ka3D). In this study, we used liposome sedimentation assays to investigate how the binding events between specific components of the CME pathway are altered by membrane localization. For our assays, we used both native (AP-2 and Clathrin) and recombinant (Epsin and AP180) proteins purified in our laboratory. The Ka3Dvalues of lipid-protein interactions were characterized using 1% PI(4,5)P2 containing liposomes via one-stage liposome sedimentation assays. Protein-protein interactions were investigated via two-stage liposome sedimentation assays. Ka3D values obtained by quantifying the amount of protein bound to the liposomes via western blotting were then used to calculate Ka2D using the above mentioned theory. Our study sheds light on how developing accurate quantitative models of assembly dynamics helps us better understand how membrane localization regulates protein assembly.