Assembly of Endocytosis-Associated Proteins on Liposomes

Abstract

This chapter describes the methodology of a cell-free system utilizing liposomes and cytosol to study the assembly reactions of proteins involved in clathrin-mediated endocytosis. Liposomes, when incubated with brain cytosol and nucleotides, undergo morphological changes and fragmentation into smaller structures that resemble closely intermediates of clathrin-mediated endocytosis. The biochemical and biophysical analysis of these reactions show that the elevated levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 , or PIP 2 ] correlate with increased recruitment of clathrin and clathrin adaptors to the liposomal surface. Inclusion into the liposomes of a surface-exposed recombinant fragment of synaptotagmin enhances the recruitment of clathrin coat proteins, thereby demonstrating the synergistic actions of proteins and lipids. Three modes of analysis are applied in the study described in the chapter: (1) observation of liposomal structure by electron microscopy, (2) protein biochemistry to assess the amounts of endocytotic proteins recruited to the liposomal surface, and (3) lipid biochemistry to measure the metabolism of phosphoinositides during the incubation reaction. Functionalized liposomes, such as the proteoliposomes, which areideally suited to investigate the synergistic effect of membrane lipids and membrane proteins on coat structure and function, are also described.