Methods

Giant Unilamellar Vesicles
GUVs were produced by a modification of the electroformation method24. The ‘Major-Minor mix' of lipids8 was prepared, consisting of the phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, 51mol%), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 22 mol%), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], 8 mol%), DOPA (1,2-dioleoyl-sn-glycero-3-phosphate, 5 mol%), soy PI (L-α-phosphatidylinositol, 8 mol%), porcine brain PI4P (L-α-phosphatidylinositol-4-phosphate, 2.2 mol%), porcine brain PI(4,5)P2 (L-α-phosphatidylinositol-4,5-bisphosphate, 0.8 mol%), CDP-DAG (1,2-dioleoyl-sn-glycero-3-(cytidine diphosphate), 2 mol%; all lipids from Avanti Polar Lipids, Alabaster, AL), fluorescent lipid analog (1 mol%, see below) and ergosterol (Sigma, St. Louis, MO; 20% ergosterol by weight, 80% phospholipids by weight). As fluorescent lipid analogs, DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, Invitrogen, Carlsbad, CA; for single-color confocal microscopy), DiDC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate, Invitrogen; for two-color confocal microscopy) and Texas-Red DHPE (Texas Red-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, Invitrogen) were used.
Lipids were combined in chloroform:methanol (2∶1, volume ratio) at 10 mg/ml total concentration and dried as a thin layer on indium-tin-oxide coated glass slides (Delta Technologies, Stillwater, MN). The slides were assembled to form a chamber by using 3 mm thick silicon spacers and holding the slides together with office clips. The chamber was filled with sucrose solution (540 mOsmol/kg) and electroformation was performed overnight using an approximately 3 Volt, 10 Hz sinusoidal voltage and copper conductive tape (3M, SPI supplies) for connections. The GUVs were harvested by sedimentation in an iso-osmolar glucose solution at 4°C.

COPII proteins
Yeast COPII proteins were expressed and purified as previously described22526272829. The H77L mutation was introduced into Sar1p by site-directed mutagenesis with Pfu polymerase (Stratagene) using the coding sequence of Sar1p in the vector pTY4028. Correct DNA sequences were confirmed by sequencing. Green fluorescent Sec13/31 protein was produced by reacting purified Sec13/31p with a 20-fold excess of Alexa 488 maleimide (Invitrogen), followed by purification on a size exclusion column. Alexa 488 labeled Sec13/31p was mixed at 40∶60 molar ratio with unlabeled Sec13/31p.

In vitro reconstitution
The purified proteins were mixed at the indicated final concentrations with HKM buffer (20 mM HEPES, pH 6.8, 160 mM potassium acetate, 1 mM magnesium chloride, final concentrations). Nucleotide exchange was accelerated either by chelating Mg2+ with 2.5 mM EDTA (Fig. 2b,d,e, 3a–h,j), or by the addition of Sec12ΔCp (Fig. 2c,f,g,j, 3k). Glycerol was used at variable concentrations to obtain a final osmolarity of approx. 600 mOsmol/kg. Concentrated GUVs were added to the osmolarity-adjusted protein solutions at 1∶20 (vol∶vol) dilution. Incubations of GUVs with COPII proteins were performed at room temperature (≈23°C).

Microscopy
Confocal microscopy was performed on a Zeiss LIVE upright microscope and on a Zeiss LSM 710 inverted microscope (Carl Zeiss Microimaging, Jena, Germany), using 40× water immersion C-Apochromat objectives, with an NA of 1.1 and 1.2, respectively. Imaging chambers consisted of coverslips coated with gelatin (Sigma), which were held together and sealed by double-sided tape.
For cryo-EM, samples were incubated directly on holey carbon grids (Fig. 3d,e,h) or incubated in a tube and pipetted onto a grid (Fig. 3a,b,f,g). Samples were vitrified by plunge freezing the grid. Imaging was performed on a Tecnai F30 electron microscope (FEI, Eindhoven, Netherlands), equipped with a 4K FEI Eagle camera. The microscope was operated at 300 kV, with a magnification of 20,000 ×, giving a pixel size of 0.59 nm at the specimen level. Data was collected at −4 µm defocus with an electron dose of 15–25 e−Å−2. The lighter areas in Fig. 3a,d–h represent the 2 µm diameter holes in the carbon support.
For negative staining, samples were incubated in a tube and spread onto a nickel grid coated with Formvar (Plano, Wetzlar, Germany). Excess liquid was blotted off with filter paper, the grid was stained with 1 % aqueous uranyl acetate and excess staining solution was blotted off. Dried specimens were examined on a Zeiss EM 900 transmission electron microscope.
The thin section electron microscopy image of a fibroblast cell (Fig. 3i) was obtained as described in14 and kindly provided by Lelio Orci (Faculty of Medicine, University of Geneva).