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

newly synthesized Gml yielded a single species which migrated more slowly on SDS-PAGE than the VSV G proteolyric fragment. Thus, even before oligomers are formed, the tail of Gml is less accessible to trypsin than that of VSV G. Interestingly, when Gml was digested after a 60-rain chase, there was a large reduction in the amount of SDS-resistant oligomer migrating at the top of the stacking gel on SDS-PAGE (Fig. 8 B, compare lanes 3 and 4) . Furthermore, we saw a second, larger band in addition to the one detected immediately after the pulse label. Both bands were endoglycosidase H-sensitive, indicating that the proteins they represent had not passed through the medial Golgi (not shown). In more detailed kinetic experiments, the amount of this upper band increased with longer chase times, with a concomitant decrease in the amount of the lower band (not shown). Based on the potential trypsin cleavage sites in the Figure 10 . Trypsin treatment of Gml and VSV G recovered after treatment with cytochalasin D. HeLa cells expressing Gml or VSV G were treated with 100 #M cytochalasin D starting 1 h before radiolabeling. Because fewer microsomes were recovered from cytochalasin D-treated cells than from untreated ceils, treated cells were radiolabeled with twice the usual concentration of radioactivity. Microsomes were prepared after a 15-min pulse and a 45-min chase, and aliquots were mock treated or treated with TPCK-trypsin as described in Materials and Methods. Gml and VSV G were immunoprecipitated using anti-VSV antibody and analyzed by SDS-PAGE. Cytochalasin D blocked formation of SDS-resistant oligomers and accumulation of the larger trypsin-cleaved band.