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

Pulse-chase studies revealed the relationship between HBsAg lipoprotein particle assembly and disulphide bond formation. We interpreted our results in terms of two classes of disulphide bonds in HBsAg, dimer crosslinks and oligomer crosslinks. Disulphide-linked dimers are formed early and are converted slowly into high molecular weight, disulphide-linked oligomers. The observation that the peak of dimer concentration (1 h) precedes the half-time for particle budding and secretion (2 h), indicates that disulphidelinked oligomers are an intermediate in the HBsAg assembly, Pulse-chase experiments in the presence of BfA showed that newly synthesized monomers were converted into a stable population of dimers. Dimer crosslinks were formed efficiently in the ER while oligomer crosslink formation was Figure 8 . Effect of Brefeldin A tre, atment on I-IBsAg distribution in SV24 cells. Indirect double immunofluorescence of HBsAg (a, rhodamine) and PDI (b, labeled with 11)3 followed by fluorescein-tabeled second antibody) on BfA-treated SV24 ceils shows that Brefeldin A does not cause HBsAg to recycle back to the ER, indicating that the HBsAg maturation compartment is distinct from the Golgi. A region in which HBsAg is concentrated and from which PDI is excluded has been marked by an arrow, c shows a pulse-chase of SV24 cells in the presence of BfA. d shows the quantitation of the experiment depicted in c with the total monomers (--~-) and dimers (-e-) seen in the nonreduced lanes represented as a percentage of total immunoprecipitable HBsAg. Note that disulphide crosslinking halts at the stage of dimer formation. Bar, 10 t~m.