Supporting InformationSchindler and and Schekman 10.1073/pnas.0910342106SI MethodsIn Vitro Budding Assay. CHO-ATF6 cells were permeabilized with40 �g/mL digitonin for 5 min, washed, and incubated in B88buffer (20 mM Hepes, pH 7.4/150 mM KOAc/5 mM MgOAc/250mM sorbitol) with 4 mg/mL rat liver cytosol, 1 mM ATP, 50 �MGDP-mannose, 40 mM creatine phosphate, and 200 �g/mLcreatine phosophokinase. Rat liver cytosol was harvested asdescribed in ref. 1, except DTT was excluded. For COPII-onlyassays, cells were treated with 1 M KoAc in B88 for 15 min onice before the reaction, GTP was raised to 300 �M, and theamount of donor membranes raised from 40 to 100 �g.

Prebudding Assay. Reactions were performed in 150 �L of bufferE [50 mM Hepes, pH 7.2/70 mM KOAc/5 mM EGTA/2.5 mMMg(OAc)2/250 mM sorbitol] with 500 �M GTP, 10 �g purifiedSec23/24, and 5 �g GST-Sar1. Permeabilized cells were incu-bated for 20 min at 30 °C, washed in buffer E, and lysed in 300�L lysis buffer (0.5% digitonin/50 mM Hepes, pH 7.2/150 mMKCl/1 mM MgCl2/0.5 mg BSA) for 1 h at 4 °C with samplerotation. Lysates were cleared with unconjugated SepharoseCL-6B beads (Sigma) for 30 min, and bound for 1 h to gluta-

thione Sepharose 4B beads (Amersham). Beads were washed 3�with lysis buffer. Sample buffer was added, beads were heated inboiling water for 5 min, and aliquots evaluated by SDS/PAGE.

Fluorescence Microscopy. Cells were grown on coverslips in a24-well plate to 50–60% confluency and toxins added forindicated times. After treatment, cells were fixed with 4%paraformaldehyde and permeabilized with 100% methanol.Antibodies were added in 0.3% BSA for 1 h. Secondary �-FITCand �-TRITC antibodies were from Jackson Immunoresearch.Cells were imaged with a Zeiss AxioObserver Z1 fluorescentmicroscope, and images captured with Metamorph software(Molecular Devices). Merges of images were performed withPhotoshop (Adobe).

Protein Purification. Human GST-Sar1 WT, H79G, and T39Nwere purified from Escherichia coli as described in ref. 1.FLAG-Sec23/His-Sec24 and HA-Sec13/His-Sec31 complexeswere purified using Ni-NTA affinity chromatography as de-scribed in ref 1. For prebudding assays, purified Sec23/24 wasexchanged with buffer E using NAP columns (GE Healthcare).

1. Kim J, Hamamoto S, Ravazzola M, Orci L, Schekman R (2005) Uncoupled packaging ofamyloid precursor protein and presenilin 1 into coat protein complex II vesicles. J BiolChem 280:7758–7768.

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Fig. S1. CHO-ATF6 cells relocalize ATF6 during ER stress. Indirect immunofluorescence of CHO-ATF6 treated for indicated times with different toxins. Green,�-FLAG (ATF6); red, �-Gos28 golgi marker; blue, Dapi.

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Fig. S2. ATF6 is not cleaved in the budding assay. CHO-ATF6 cells were analyzed in a budding reaction that blotted for full-length ATF6. Because cleaved ATF6is a soluble protein, a 15% fraction of the supernatant was also examined. CHO.K1 cells were analyzed to control for proteins in the cytosol that may cross-reactwith �-FLAG antibodies.

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