Supplementary materials Detailed methods Cell culture and drug treatment. HEK 293 cells were cultured in DMEM (Gibco-BRL) supplemented with 10% fetal bovine serum. To inhibit glucosidase Ι and ΙΙ, castanospermine (Sigma) was added to a final concentration of 1 mm 1 h prior to pulselabeling. The proteasome inhibitor, lactacystin (Kyowa medics Co.), was added to the culture medium to a final concentration of 1 mm 4 h prior to pulse labeling. The inhibitors were also present in the medium during the pulse and chase periods. To inhibit glucosidase ΙΙ, N-butyl-deoxynojirimysin (Wako) was added to a final concentration of 1 mm at the time point of chase 5 min. Plasmid construction and transfection. EDEM-HA, A1AT (NHK), CNX-HA, and CRT/CNXtm were constructed as previously described (1, 2). FS-CNX was a kind gift from Dr. D. B. Williams (3). EDEM-myc was constructed by subcloning EDEM-HA into the Kpn Ι and Xho Ι sites of the pcdna3.1/myc-his(+)a vector (Invitrogen). Using PCR, the HA tag and TGA of EDEM-HA were deleted and a Xho Ι site was introduced at the C- terminus. CNX/HAtm was constructed as follows: the transmembrane region of
haemagglutinin was fused to FS-CNX followed by the HA tag and KKXX. The membrane region of HA was subcloned into the Dsa Ι and BamH Ι sites of FS-CNX. FS-CNX+HAtm was subcloned into the Hind ΙΙΙ and EcoR Ι sites of pcdna3.1, and the HA tag (YPYDVPDYA) and a retention signal (DEKKMQ) were introduced by PCR. The Fugene6 transfection reagent (Roche) was used for plasmid transfections 36 h prior to performing the pulse-chase assays. Metabolic labeling, immunoprecipitation and immunoblotting. Cells were labeled with EXPRESS 35 S protein labeling mixture (NEN) in methionine-free medium (Gibco-BRL), and chased in medium containing excess methionine. Cells were lysed in a buffer containing 1% NP-40, 150 mm NaCl, 50 mm Tris-Cl (ph 8.0) and protease inhibitors (1 mm N-methyl maleimide, 1 mm phenylmethylsulphonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A). Lysates were immunoprecipitated using appropriate antibodies and coupled to protein A or G sepharose beads (Amersham Pharmacia). The beads were washed in high ionic extraction buffer (1% NP-40, 400 mm NaCl, 50 mm Tris-Cl (ph 8.0)) twice, and in 10 mm Tris-Cl (ph 7.4), eluted in Laemmli buffer, and resolved by SDS-PAGE. Gels were fixed with 50% TCA, washed, and enhanced with 1 M sodium salicylate. Radioactivity was quantified by exposing the gels to a phosphoimager (Strom, Amersham
Pharmacia) and the gels afterward were exposed to X-ray films. For western blotting, proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes followed by ECL (Amersham Pharmacia). Antibodies. For immunoprecipitation, antibodies specific for CNX (Stressgen SPA-860), CRT (Stressgen and ABR), HA (SantaCruz), A1AT (Dako), and EDEM antibody were used. Anti-EDEM antibodies were generated by immunizing rabbits with a synthetic peptide (ERDQEEKFVHRPKSQE). Western blotting was performed using antibodies to myc (9E10, SantaCruz) and CNX antibody (Stressgen SPA-860).
References 1. N. Hosokawa et al., EMBO Rep. 2, 415 (2001). 2. I. Wada, S. Imai, M. Kai, F. Sakane, H. Kanoh, J. Biol. Chem. 270, 20298 (1995). 3. U. G. Danilczk, M. F. Cohen-Doyle, D. B. Williams, J. Biol. Chem. 275, 13089 (2000). 4. J. Wang, A. L. White, Biochemistry 39, 8993 (2000) 5. K.S. Cannon, A. Helenius J. Biol. Chem. 274, 7537 (1999) Supplemetal figure legends Supplemental Figure 1. The effects of overexpressing CNX. HEK 293 cells were pulselabeled for 15 min and chased the periods indicated, immunoprecipitation was performed with anti-a1at antibody. Left panel: Cells were transfected with NHK or co-transfected with NHK and CNX-HA. Right panel: Cells were co-transfected with NHK and EDEMmyc or with NHK, EDEM-myc, and CNX-HA. Supplemental Figure 2. Post-translational inhibition of NHK glucose trimming retarded degradation. HEK 293 cells were transfected with NHK (supplementary Fig. 2A) or co-
transfected with NHK and EDEM (supplementary Fig. 2B), pulse-labeled for 15 min with [ 35 S]-methionine, and chased for the periods indicated. To inhibit trimming the Glu 1 into the Glu 0 form of NHK by glucosidase ΙΙ, cells were treated with N-butyl-deoxynojirimysin (BDNM) after 15 min pulse in the absence of the drug. Under this condition, release of NHK from CNX was prevented (4, 5).