PC1 is exported out of the ER in large COPII-coated vesicles. (A) A representative field of COPII vesicles isolated from cell-free vesicle budding reaction visualized by SIM. One (indicated by the arrow) contained PC1-GFP (green) that was entirely encapsulated by the COPII inner coat proteins SEC23A/24D-647 (magenta). The magnified xz and yz views are displayed underneath the overview. Approximately 20 images were collected from each experiment with fluorescence controls. n = 3. Bar, 200 nm. (B) Relative size of COPII carriers of PC1 compared with regular COPII vesicles quantified by flow cytometry; numbers in graphs represent the percentage of particles in the respective subpopulation. (i) Singlets were plotted for the fluorescence intensities of the COPII coat proteins SEC23A/24D-647 (APC-A) and the lipid dye FM4-64 (PE-Cy5-A). (ii) Particles positive for both COPII and lipid are COPII vesicles and were plotted for the fluorescence intensity of PC1-GFP (FITC-A). (iii and iv) The values of side scatter (SSC-A) versus forward scatter (FSC-A) of COPII vesicles that are PC1-GFP negative (iii) or GFP positive (iv) are plotted. n = 3.