The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre- Golgi compartment

Subconlluent monolayers in 35-mm dishes were washed (two times) in PBS, incubated in cysteine-free MEM/10% DFBS for 30 rain at 37~ and pulse labeled with 100-200 #Ci/ml [35S]cysteine for 30-60 min. Post pulse the cells were incubated with complete media containing 25 x excess unlabeled cysteine. For chloroquine experiments, only the chase medium contained chloroquine (100 #M). Labeling with 3H- [9, 10] palmitate (250/~Ci/ml) was for 6 h at 37~ in complete medium. Cells were washed (three times) with cold PBS and lysed in 150 mM NaCI, 50 mM Tris-HC1 (pH 8.0), 1% NP-40, 0.5 % sodium deoxycholate, 0.1% S DS, and 1 #g/ml of chymostatin, leupeptin, antipain, and pepstatin A by incubation on ice for 15 rain. The lysates were centrifuged for 3 min in a microfuge, and the supernatants were incubated with protein A/G agarose beads coated with human anti-RV serum at 4~ for at least 2 h on a rotating device. Immunoprecipitates were washed (three times) with RIPA buffer (50 mM Tris-C1, pH 7.5, 150 mM NaC1, 1% NP-40, 0.5 % sodium deoxycholate, 0.1% SDS) and once with water. Proteins were eluted by heating at 100~ in SDS-gel sample buffer (50 mM Tris-HC1, pH 6.8, 2% SDS, 2% B-mercaptoethanul, 10% glycerol, 0.1% bromophenol blue).