In Vitro Reconstitution of Plant COPII Vesicles.

In vitro reconstitution studies enable the controllable and stepwise investigation of complicated biochemical processes. In yeast and mammals, in vitro reconstitution of COPII vesicles marked a pivotal point in characterizing the endoplasmic reticulum-to-Golgi anterograde trafficking route and revealed how vesicles mediate the selective and reliable transportation among topologically equivalent compartments. By providing the necessary physiological conditions in a cell-free environment, it enables the dissection of essential components required for the vesicle formation. To enrich and purify the small amount in vivo membrane-bounded compartments, it simplifies the evaluation of vesicle regulation by distinct external stimuli or upstream signals. Here, we describe the preparation of plant microsomes and cytosol for the reconstitution of plant COPII vesicles. Purified vesicles can be used for further biochemical or microscopical analyses.