Vesicle budding from endoplasmic reticulum

This chapter discusses the process of vesicle budding from endoplasmic reticulum (ER). Protein transport along the secretory pathway has historically been evaluated by molecular characterization of covalent modifications on transported proteins, lipids, and carbohydrates and by the fractionation and evaluation of membrane compartments. This chapter details the procedures necessary for evaluating the polypeptide translocation and protein sorting into transport vesicles using purified components. Cell-free reactions that reproduce transport steps between organelles in the secretory pathway are used to identify the proteins involved in particular steps and mechanistic aspects of this process. Secretory proteins comprising soluble and membrane-bound cargo molecules are assembled in the ER and sorted from resident ER proteins by their capture into transport vesicles that are directed to the Golgi apparatus. The chapter summarizes the current protocols for the preparation of donor ER membranes and procedures for the isolation of the cytosolic coat components. The chapter concludes with a discussion of in vitro biochemical analysis of vesicle formation and the packaging of soluble and membrane proteins into COPII vesicles.