Cell-free Generation of COPII-coated Procollagen I Carriers

Abstract

The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the isolation and detection of reconstituted COPII-coated carriers. This cell-free reaction can be used to test conditions that stimulate or suppress the packaging of PC1 into COPII-coated carriers.

Keywords: Budding; COPII; Collagen; Membrane; Reconstitution.

Figure 1.. Schematic overview of the experimental procedure.

The preparations of cytosol and donor membrane for the budding reaction were described in Procedure A and Procedure B, respectively. The assembly of budding reactions and isolation of vesicles from budding reactions were described in Procedure C. Packaging efficiency of COPII cargos were assessed by immunoblotting as described in Procedure D (Republished from Gorur et al., 2017 with modifications).

Figure 2.. COPII is required to package PC1 into reconstituted vesicles.

Budding requirements of PC1 and HSP47 (a collagen-specific chaperone) were assessed under different incubation conditions described in Table 1. The top fraction after flotation was taken from each sample and analyzed by immunoblotting. Ribophorin I is an ER resident protein that serves as a negative control. ERGIC53 and SEC22B are found in conventional COPII vesicles and serve as positive controls. (Republished from Gorur et al., 2017 )