In vitro COPII vesicle budding assay

Purification of COPII coat components Sar1, Sec23/24, and Sec13/31, and the vesicle budding assay were performed as previously described⁷⁰. Microsomal membranes were prepared from yeast cells expressing Ehg1-3HA present in a high-copy plasmid. Frozen microsomal membranes were thawed and washed once with 0.5 M NaCl in buffer 88 (B88, 20 mM HEPES, 150 mM KCl, 5 mM Mg (CH₃COO)₂, 250 mM Sorbitol) and then twice with B88 containing protease inhibitors. The 200-µl reaction mixtures containing washed membranes (250 µg/mL), ATP plus ATP regeneration system, and GTP were incubated for 30 min at 25 °C in the presence or absence of the purified COPII coat components. After the incubation, aliquots representing the total membranes were transferred to fresh tubes. The remaining reactions were centrifuged at 10,000 × g for 5 min to separate the middle speed supernatant (MSS), which contained generated COPII vesicles from the heavy donor membranes. The MSS was further centrifuged at 100,000 × g for 1 h to collect the COPII vesicles as pellets. The total membrane fractions and COPII pellets were analyzed using SDS-PAGE and immunoblotting with anti-HA monoclonal antibody (Millipore Sigma, St. Louis, MO, USA). Additionally, Erv46 and Sec61 were considered as positive and negative controls, respectively, to determine the efficiency of the budding reaction. Signals were visualized and quantified using the LI-COR Odyssey system (LI-COR, Inc., Nebraska, USA). A percentage of each protein incorporated in the COPII vesicle fraction was compared with the total amount of each protein present in the reaction mixture. The values were plotted as packaging efficiency. Each set of reactions was performed in triplicates. Anti-Erv46 antiserum was a kind gift from Charles Barlowe (Dartmouth Medical School, USA). Anti-Sec61 antiserum was generated by injecting rabbits with a chemically synthesized Sec61 peptide coupled with keyhole limpet hemocyanin as previously reported⁷¹.