Generation of multibudded structures by COPII, revealed by EM.
Black scale bars = 200 nm; white = 100 nm. (a–h) Cryo-EM view of membrane extensions generated by COPII [2µM Sar1p, 320nM Sec23/24p, 520nM Sec13/31p, 2.5mM EDTA, 1mM GMP-PNP]. (a) Overview and (b) magnified inset. Tubular extensions were coated (arrows in panel b) and carried constrictions at ≈100 nanometer spacing. (c) Schematic view and model: Drawing of a COPII vesicle string based on cryo-EM. Red denotes the lipid bilayer, green the protein coat. The COPII coat is rigid and may act like an exoskeleton. Bottom: Multibudded vesicles showed elongated shapes. A propensity of the COPII coat to form spheres may contribute to fission at the necks. (d,e) Both large and small vesicles were coated (d,e: arrows), but some large vesicles remained uncoated (e: black arrowhead). An internal vesicle (d: white arrowhead) remained uncoated. (f) Network of multibudded tubules. Occasional branches are seen in (a) and (f) [gray arrow]. Clover-shapes constituted cross-over tubules. Vesicle strings were apparently well-preserved on the carbon support, but often ended at the holes through which the solution had been blotted (white arrowheads). (g) Reconstituted vesicle strings suggest a mechanism for transport of large cargo. Hypothetical cargo is depicted as boxes. A 300 nm long procollagen bundle, for instance, may occupy three consecutive buds, with fission occurring at the adjoining necks. (h) Some vesicle strings were still connected to a larger liposome and appeared similar to ER exit sites in cells. (i) EM of ER exit sites in a wild type fibroblast cell showed multibudded tubules with a striking resemblance to those produced in vitro from artificial liposomes. (j) Multibudded tubules (including occasional branches) in reconstituted samples were also revealed by negative stain EM. The inset shows a multibudded tubule emanating from a collapsed liposome [2µM Sar1p, 320nM Sec23/24p, 520nM Sec13/31p, 2.5mM EDTA, 1mM GMP-PNP]. (k) The use of the GTP-locked Sar1p(H77L) mutant produced multi-budded tubules in the presence of hydrolysable GTP (negative stain; same sample as in Fig. 2j).