Oligomerization of a membrane protein correlates with its retention in the Golgi complex

Cells expressing VSV G or mutant G proteins were rinsed once with PBS, starved for 15 rain in methionine-free DME, and labeled for the indicated times with 100 #Ci/ml Trans35S-Label (ICN Radiochemicals, Irvine, CA) or L-[35S] in vitro cell labeling mix (Amersham Corp., Arlington Heights, IL) in serum-free, methionine-free DME. Cells were solubilized immediately or after the appropriate chase period in serum-free DME containing a threefold excess of unlabeled methionine. Cells were lysed in detergent solution (50 mM Tris, pH 8.0, 1% NP-40, 0.4% deoxycholate, 62.5 mM EDTA, and 0.13 TIU/ml aprotinin). Samples were immunoprecipitated using a polyclonal anti-VSV antibody (produced by immunization of rabbits with purified VSV) and fixed Staphylococcus aureus (Calbiochem-Behring Corp., San Diego, CA). After heating to 100*C for 3 rain in Laemmli sample buffer containing 5 % fl-mercaptoethanol, samples were electrophoresed on 10% SDS-polyacrylamide gels as described (Laemmli, 1970) . Marker proteins were 14C-methylated standard molecular weight markers (Amersham Corp.). Labeled proteins were detected by fluorography (Bonner and Laskey, 1974) . am chimeras were radiolabeled and solubilized as above but immunoprecipitated using anti-human chorionic gonadotropin (hCG) antibody (Organon Teknika-Cappel, West Chester, PA) and electrophoresed on 12 % SDS-pelyacrylamide gels.