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

Intact cells were treated with the membrane-permeable, thiol-cleavable cross-linker DSP to determine if E1 was arranged in noncovalent aggregates. A 60-rain labeling period was used to ensure that endogenous proteins that might be associated with E1 were labeled as well. When immunoprecipitates were separated by SDS-PAGE, slightly less monomeric and dimeric E1 was seen after cross-linking due to formarion of larger oligomers with an apparent molecular weight >200 kD (Fig. 5 A, arrowhead) . Crosslinking efficiency in intact cells was not improved substantially by increasing the concentration of DSP up to 1 mg/ml (not shown). Cross-linking efficiency was improved greatly by adding DSP to radiolabeled cells at the time of lysis. Cells were lysed in the presence of the nonionic detergent NP-40 (without SDS) so as not to disrupt protein-protein interactions. Under these conditions, cross-linking efficiency was much greater as very little monomeric E1 remained; however, no large aggregates were seen at the top of the resolving blocked in a pre-Golgi compartment. Subconttuent monolayers were pulse labeled with [35S]cysteine for 30 min and then chased in complete media containing excess unlabeled cysteine for the indicated time periods (h). Cells were lysed and E1 was recovered from the postnuclear supernatants by incubation with human anti-RV serum and Omnibind agarose beads. Samples were divided into three aliquots and treated with or without endo H (H lanes) or endo D (D lanes) or incubated without enzyme ( -lanes). The proteins were separated by SDS-PAGE on 10% polyacrylamide gels and fluorographed. Golgi-specific processing of E1 glycans is indicated by an arrowhead. 14Clabeled protein standards (kD) are included for reference. In the endo D lanes the E1 bands appeared to be compressed in the chased samples. This flattening effect on E1 was most likely due to BSA (in the endo D preparation) which migrates slightly above El. gel (Fig. 5 B, -f3ME) . Rather, the E1 monomers, dimers, and trimers were cross-linked into oligomers >200 kD that possibly represent pentamers and hexamers (Fig. 5 B, arrowheads) . These oligomers presumably consisted mainly of El, since no other proteins including BiP (78 kD) specific to the cross-linked samples were evident after cleaving the crosslinker with /3-mercaptoethanol (Fig. 5 B, +BME) . Since CHO BiP can be radiolabeled with cysteine and contains 60 lysine residues (59) (some of which would be expected to be available for cross-linking with DSP), we would expect to be able to detect BiP under these conditions if it were associated with El. Furthermore, E1 from CHOE1 cells was recognized by four different mAbs which specify important immunologic epitopes, suggesting that E1 is properly folded (not shown).