SUPPLEMENTARY INFORMATION

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1 Supplementary Methods Protein expression and in vitro binding studies. Recombinant baculovirus carrying GST, GST-mCC, GST-mSec, GST-mSecA and GST-mSecD were generated according to the manufacturer s instructions (BD Biosciences, CA). Sf cells grown in suspension were infected with baculovirus for days and then the cells were harvested and resuspended in cold buffer containing 0 mm Tris (ph.), 00 mm NaCl, 0. % Triton X-00, 0. % sodium deoxycholate, 0. mm β-mercaptoethanol plus protease inhibitors. The lysates were centrifuged at,000 g for min. Glutathione Sepharose beads were added to the supernatants and incubated for h at C. The beads were washed in buffer (0 mm Tris (ph.), 00 mm NaCl) and stored on ice. Bacteria overexpressing full length His -mbet were sonicated in buffer containing 0 mm Tris (ph.0), 0 mm NaCl, 0 mm immidazole, mm β- mercaptoethanol, and protease inhibitors. The lysate was centrifuged at,000 g for 0 min at C and the supernatant was treated to a Ni + -NTA column at C. Bound His - mbet was eluted with buffer containing 0 mm Tris (ph.0), 0 mm NaCl, 00 mm immidazole, mm β-mercaptoethanol and protease inhibitors. Eluted His -mbet was dialysed overnight against buffer (0 mm Tris (ph.0), 0 mm NaCl, and mm β- mercaptoethanol). For in vitro bindings, a 00 µl binding reaction containing His -mbet was incubated overnight ( C) with Glutathione Sepharose beads containing 0. µm GST or GST fusion protein in binding buffer (0 mm Tris (ph.), 00 mm NaCl, mg/ml BSA, 0. % Triton X-00, 0. % NP-0). The beads were then centrifuged at,000 g for min and washed times with buffer containing 0 mm Tris (ph.) and 00 mm

2 NaCl. At the end of the last wash, the remaining liquid was aspirated from the beads and µl of X SDS sample buffer was added to the beads (final volume approximately 0 µl). Samples (0 µl) were subjected to SDS-PAGE and immunoblotted using the ECL method. Approximately -.% of the mbet bound to msec depending on the preparation. Immunopreciptation. Sixteen h after transfection with pcmv-myc or pcmv-mycmbet, approximately 0 COS- cells were trypsinized and harvested. Cell pellets were resuspended in 0.ml of lysis buffer (0 mm Tris (ph.), 00 mm NaCl and protease inhibitor cocktail). Lysates were generated by sonicating the cell suspension for sec with a microtip. After sonication, 0. % NP0 was added to lysates before they were incubated for 0 min at C. The lysates were clarified by centrifugation at,000 rpm for min at C. Anti-myc monoclonal antibody (E0) was added to the supernatants and incubated overnight at C. The next day, 0 µl of Protein A-Sepharose beads were added to the samples and incubated for h. The beads were centrifuged at,000 rpm ( C) and washed times in lysis buffer. The immunoprecipitates were subjected to SDS- PAGE and immunoblotted. Preparation of yeast lysates used for in vitro binding studies. To prepare yeast lysates used for the in vitro binding studies in Fig. Sb, 00 OD units of cells were resuspended in ml of lysis buffer (PBS, mm DTT plus protease inhibitors). An equal volume of acid-washed glass beads was added to the cells and the sample was vortexed three times. Each round of vortexing lasted min and between rounds of vortexing the sample was

3 placed on ice. Triton X-00 was added to the lysate to a final concentration of % before the lysate was clarified by centrifugation at,000 g for 0 min. The protein concentration of the supernatant was measured using the Bradford assay. Homotypic COPII vesicle tethering assay. The homotypic tethering assay was performed as described before. Co-isolated vesicles containing VSV-G* were quantitated by autoradiography. As a no vesicle control, vesicle release reactions were performed in the presence of GDP-locked Sar TN (00 nm) to block budding. For the untagged control, radiolabeled VSV-G*-containing vesicles were combined with COPII vesicles from nontransfected cells (no VSV-G). In Fig. b (inset) and d, the vesicles were immuno-isolated with α-myc and analyzed by immunoblotting. To estimate the relative abundance of msec on the co-isolated vesicles, we divided the relative signal for msec by the relative signal for the cargo marker VSV- G-myc on the gel shown in Fig. d. To determine the significance of the abundance of msec relative to maximally coated vesicles, the value obtained from the co-isolated vesicle fraction formed in the presence of GTP was expressed as a percentage of the value obtained from the co-isolated vesicle fraction formed in the presence of GMP-PNP. Vesicles formed in the presence of GMP-PNP is 00%. In vitro vesicle binding and tethering assay with yeast COPII vesicles. Vesicle binding experiments were performed as described previously with some exceptions. A typical binding reaction contained a 0 µl slurry of ytrappi (Betp tagged with Protein A) containing IgG Sepharose beads and 00µl of supernatant from one vesicle

4 budding reaction. To test if the interaction between Secp and Betp is required for the binding of COPII vesicles, increasing concentrations of bacterially expressed fusion protein was incubated with vesicles and ytrappi for h at C. To determine if sec- mutant vesicles are defective in binding, vesicles were generated from wild type or mutant cells at C, and incubated with ytrappi containing beads at C or C for 0 min. To determine if vesicles formed in the presence of GTPγS bind ytrappi, budding reactions were performed for 0 min (instead of 0 min) in the presence of 00 µm GTPγS. The longer incubation time compensates for the reduction in vesicle budding caused by GTPγS. Although binding was not detected before, increased vesicle yields in combination with the larger amounts of ytrappi used in this study, enabled us to detect binding. All binding reactions were normalized to ConA precipitable counts. Vesicle tethering experiments were performed as before. All fusion proteins were added to the assay at. µm. Preparation of cytosol free vesicles. A total of 0- vesicle budding reactions were performed for 0 min in the presence of GTP and 0 min in the presence of GTPγS. Donor cells were pelleted, and the vesicles released into the supernatant were fractionated from cytosol on a Sephacryl S-00 column. The peak vesicle fractions were pooled and centrifuged at 00,000g for 0 min. The supernatant was removed and the pellet was resuspended in transport buffer (mm Hepes ph.., mm potassium acetate,.mm magnesium acetate, 0.M sorbitol, x protease inhibitor cocktail) for vesicle binding assays, or dissolved in SDS sample buffer for Western blot analysis. GST and GST-Secp were added to the vesicle binding assay at. µm.

5 Preparation of vesicles stripped of COPII coats. COPII coats were removed from vesicles by centrifugation through a 0.M sorbitol cushion prepared in transport buffer (mm Hepes ph.., mm potassium acetate,.mm magnesium acetate, 0.M sorbitol, x protease inhibitor cocktail). The vesicle pellet was resuspended with transport buffer for vesicle binding assays, or dissolved in SDS sample buffer for Western blot analysis. To determine if the stripped vesicles are fusion competent, the vesicle pellet was resuspended in transport buffer with an ATP-regenerating system, cytosol, Golgi membranes, and then incubated at 0 C for 0min. Cloning and expression of ytrappi subunits. The coding regions for yeast TRS, TRS, TRS, TRS0, BET and BET were PCR-amplified from yeast genomic DNA and ligated into the DUET vectors-petduet, pcoladuet and pcdfduet (Novagen). The three plasmids were co-transformed into E. coli BL(DE), and the ytrappi subunits were expressed overnight at o C by induction with 0.mM IPTG at an OD 00 of 0.. The complex was purified by Ni + -NTA affinity chromatography followed by gel filtration using a Superdex-00 column. Supplementary Reference. Ruohola, H., Kabcenell, A. K. & Ferro-Novick, S. Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec mutant. J Cell Biol 0, - ().

6 . Xu, D. & Hay, J. C. Reconstitution of COPII vesicle fusion to generate a pre- Golgi intermediate compartment. J Cell Biol, -00 (00).. Sacher, M. et al. TRAPP I implicated in the specificity of tethering in ER-to- Golgi transport. Mol Cell, - (00).. Barrowman, J., Sacher, M. & Ferro-Novick, S. TRAPP stably associates with the Golgi and is required for vesicle docking. EMBO J, - (000).. Loh, E., Peter, F., Subramaniam, V. N. & Hong, W. Mammalian Bet functions as a cytosolic factor participating in transport from the ER to the Golgi apparatus. J Cell Sci, 0- (00).. Bi, X., Corpina, R. A. & Goldberg, J. Structure of the Sec/-Sar pre-budding complex of the COPII vesicle coat. Nature, - (00).. Barlowe, C. Coupled ER to Golgi transport reconstituted with purified cytosolic proteins. J Cell Biol, 0-0 ().. Miller, E. A., Liu, Y., Barlowe, C. & Schekman, R. ER-Golgi transport defects are associated with mutations in the Sedp-binding domain of the COPII coat subunit, Secp. Mol Biol Cell, - (00).

7 a Transfection: IP (0%) Lysate (.%) pcmv-myc pcmv-myc-mbet msec b GST GST-Secp Lane: GST-Secp Input (0.%) Trsp IP (0%) Lysate (.%) mer0 GST GST-Yptp GST-Secp Input (0.%) Trsp IP (.%) Lysate (0.%) myc-mbet Lane: Lane: Supplementary Figure S. Sec interacts with Bet in mammalian cells and yeast. a, mbet co-precipitates with msec in vivo. Because our anti-mbet antibody does not precipitate efficiently, we tested for an in vivo interaction between mbet and msec by transfecting COS- cells with a plasmid that expresses tagged mbet (pcmv-myc-mbet). Lysates prepared from COS- cells transfected with plasmid pcmv-myc or pcmv-myc-mbet were precipitated with anti-myc antibody and blotted for msec, the ER protein ER0 and myc-mbet. Tags at the amino-terminus of mbet are functional. The percent of the total loaded onto the gel is indicated to the left of the lanes. Approximately.% of the msec in a lysate, but not the ER protein ER0, specifically co-precipitated with myc-mbet. b, ytrapp interacts with Secp. Beads containing µm GST (lane, top and bottom panels), GST-Secp (lane, top panel; lane, bottom panel), GST- Secp (lane, top panel) and GST-Yptp (a Rab homologue in yeast-see lane, bottom panel), were incubated with ml of yeast lysate (mg/ml) for h at C. The beads were washed times with PBS plus % Triton X-00 and protein was eluted with SDS sample buffer. The eluates were then subjected to SDS-PAGE and blotted with antibody directed against the ytrapp subunit Trsp. Lane, 0.% of the input lysate used in the binding reaction.

8 % of wild type binding Fusion Protein (µg) GST-Secp GST-Secp* GST Supplementary Figure. The GAP activity of Secp does not play a role in the binding of ytrappi to COPII vesicles. The crystal structure of Secp revealed that the insertion of Arg into the Sarp active site is important for GTPase stimulation. To determine if Arg plays a role in the binding of ytrappi to COPII vesicles, we mutated Arg in GST-Secp to an alanine (GST-Secp*) using site-directed mutagenesis. When excess soluble GST-Secp* was added to the vesicle binding assay, it efficiently inhibited the binding of ytrappi to COPII vesicles. All binding assays were performed as described in the supplementary information. Mutating Arg to alanine in Secp blocks GAP activity (Elizabeth Miller, personal communication). Error bars are SD. In all experiments n=.

9 a b vesicles+golgi 00 vesicles+golgi +GST-Secp doi: 0.0/nature0 c wild type 000 wild type 000 sec ConA precipitable counts (cpm) sec ConA precipitable counts (cpm) ConA precipitable counts (cpm) wild type 00 wild type vesicles+golgi sec- sec- 00 vesicles+golgi +GST-Secp anti-outer chain precipitable counts (cpm) anti-outer chain precipitable counts (cpm) 0 0 anti-outer chain precipitable counts (cpm) 0 Supplementary Figure. Secp, but not Secp, is required for COPII vesicle tethering. a, Excess GST-Secp blocks vesicle fusion, but not COPII vesicle tethering. Assays were performed as described in the supplementary information and in the legend to Figure a and b. Vesicles tether (top), but do not fuse (bottom), to the Golgi in the presence of excess soluble GST-Secp. Excess Secp has previously been reported to disrupt fusion, possibly by binding to the surface of membranes and blocking the, fusion machinery. b, The sec- mutant does not display a defect in tethering at C. Vesicles were formed from wild type fractions or sec- donor cells with sec- mutant cytosol for 0 min at C. The donor cells were pelleted and the supernatant was incubated with wild type or mutant Golgi membranes at C for 0 min. c, The sec- mutant is partially defective for vesicle tethering at C. All assays were the same as in b only vesicles were formed at the permissive temperature and then incubated with wild type or mutant Golgi membranes at C for 0 min. This ts defect in vesicle tethering was comparable to the vesicle binding defect observed with sec- mutant vesicles.

10 a control GTP GTPγS Secp Secp Bosp Sarp Lane: b control stripped Secp Bosp Lane: Supplementary Figure. The binding of ytrappi to COPII vesicles is dependent on the presence of the COPII coat. a, The COPII coat is locked onto vesicles in the presence of GTPγS. Cytosol was removed from vesicle fractions formed in the presence of GTP (lane ), GTPγS (lane ), or apyrase (lane, no vesicle control) as described in the supplementary information. For samples prepared in the presence of GTP and GTPγS, equivalent amounts of vesicles (equal ConA precipitable counts) were then subjected to SDS-PAGE analysis and blotted with antibodies directed against Secp, Secp, Bosp and Sarp. Sarp was enriched in vesicles formed in the presence of GTPγS (compare lanes and ). Assuming that COPII vesicles are maximally coated in the GTPγS sample, we estimated that approximately ~% of the Secp was retained on vesicles formed in the presence of GTP when Bosp was used as a marker to normalize to vesicle yield. b, Sorbitol treated vesicles lack Secp. Vesicles were mock treated (control) or treated with 0.M sorbitol (stripped vesicles), subjected to SDS polyacrylamide gel analysis and analyzed for the presence of Secp. Vesicle fractions were normalized to ConA precipitable counts and the SNARE Bosp. 0