Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment

HBsAg particles were collected from SV24 medium after 24 h radioactive labeling (100 ~Ci/mi [35S]methionine and 10% of normal cold methionine) by spinning 12 h at 34,000 rpm in a Beckman SW40Ti rotor at 4"C. The pelleted material was suspended in 100 mM potassium phosphate, pH 7.5, and divided into aliquots that were incubated 10 h at 37~ in 100 mM potassium phosphate, pH 7.5, containing 5 mM reduced glutathione, 0.1% (wt/vol) Triton X-100, and 5, 25 or 125 ~l of the lysate of the rat PDI-eDNA bearing haculovirus-infected ceils or of the wild type baculovirus infected ceils (containing 7 mg/ml protein as measured with BioRad protein assay). The total incubation volume was 1.0 mi. After the incubation, 200 t~l of 120 mM NEM was added to each sample to block the free thiols. HBsAg was immunoprecipitated as described for the pulse-chase experiments, except the final washes were omitted. Before preparing the irnmunoprecipitates for SDS-PAGE, they were washed once with RIPA-buffer and once with 10 mM Tris-Cl, pH 7.4. After the electrophoresis the gels were processed for autoradiography and quantitated as described for pulse-chase experiments.