Generation of rigid membrane extensions by COPII, visualized by confocal microscopy.
Scale bars = 10 µm. (a) Schematic view. The reaction was reconstituted in vitro by mixing purified red fluorescent GUVs and purified COPII components [2µM Sar1p, 320nM Sec23/24p, 520nM Sec13/31p; 1µM Sec12ΔCp or 2.5mM EDTA (to facilitate nucleotide exchange)]. (b) COPII produced long, rigid membrane extensions that appeared tubular at confocal microscopy resolution. (c) Green fluorescently labeled Sec13/31p showed a preferential COPII localization at highly curved tubules compared to the low curvature GUV. (d/e) Sec13/31p was essential. Extensive rigid tubulation was observed with full COPII and GMP-PNP (even at lowered COPII concentrations: 1µM Sar1p, 160nM Sec23/24p, 260nM Sec13/31p, 2.5mM EDTA, 0.1mM GMP-PNP in panel d). In contrast, in the absence of Sec13/31p, GUVs and smaller liposome material aggregated (panel e). Inset: 3D maximum intensity projection (nonlinear intensity scale). Omitting Sec13/31p from the coat caused GUVs to adhere to each other. See also Fig. S2. (f) At concentrations of only 0.2µM Sar1p [320nM Sec23/24p, 520nM Sec13/31p, 1µM Sec12ΔCp, 1mM GMP-PNP] mostly intact GUVs were observed. Close inspection revealed shorter rigid tubules emanating from the GUVs. Extensions are more easily discernible when using a nonlinear intensity scale (right). (g) The N-terminal amphipathic helix of Sar1p was essential for recruiting COPII. The truncated Δ23 mutant did not recruit COPII marked by a green labeled Sec13/31p (right) to the red labeled lipid bilayer (left) and did not induce tubulation of GUVs [2µM Δ23-Sar1p, 320nM Sec23/24p, 520nM Sec13/31p, 1µM Sec12ΔCp, 1mM GMP-PNP]. (h) With hydrolysable GTP, the COPII proteins produced a different and more heterogeneous picture consisting of intact GUVs, micron-sized vesicles and soft tubules [2µM Sar1p, 320nM Sec23/24p, 520nM Sec13/31p, 1µM Sec12ΔCp, 1mM GTP]. (i) With EDTA instead of Sec12ΔCp, GUVs appeared mostly intact, but some had a granular appearance or were surrounded by micron-sized vesicles [2µM Sar1p, 320nM Sec23/24p, 520nM Sec13/31p, 2.5mM EDTA, 1mM GTP]. (j) In contrast to panels (h) and (i), the Sar1p mutant H77L as part of the COPII coat generated long, straight, rigid extensions with hydrolysable GTP [2µM Sar1p(H77L), 320nM Sec23/24p, 520nM Sec13/31p, 1µM Sec12ΔCp, 1mM GTP].