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

We analyzed the maturation process biochemically to separate the early events occurring in the ER from the ones which occur in the PDI excluding compartment. We approached this by studying the time course of intermolecular disulphide bond formation and of particle secretion by pulsechase immunoprecipitation experiments. SV24 cells were pulsed for either 2 or 10 min with radioactive methionine and chased for various times. HBsAg was then immunoprecipitated from the cell extracts and the culture media and analyzed by SDS-PAGE in reducing (+) or nonreducing conditions ( -) (Fig. 6, A and B) . Analysis of the reduced sampies in which all of the HBsAg entered the gel and migrated as monomers (M/gp R and M/p ~ for glycosylated and non- glycosylated forms, respectively) showed that newly synthesized HBsAg was not subjected to significant proteolysis, which would have complicated the kinetic analysis. This agrees with the results of previous experiments with the SV24 cell line which showed that it secretes >95 % of the synthesized HBsAg as lipoprotein particles within 10 h (Simon et al., 1988) . The nonreduced lanes of the gels are conveniently interpreted in terms of our two class description of disulphide bonds in HBsAg. The monomer bands for the gly-cosylated (M/gp N) and the nonglycosylated (M/p N) disappear with the formation of the dimers showing the occurrence of the dimer crosslinks. The formation of oligomer crosslinks is reflected by the occurrence of material which runs at very high molecular weight or does not enter the gel in the nonreduced state.