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

Trypsinization of microsomes eliminated the SDS-resistant form of Gml. To ask if proteolysis dissociated the oligomers as well, we subjected trypsin-treated Gml-containing microsomes to sucrose gradient sedimentation. Microsomes prepared from HeLa cells after a 5-min pulse and a 60-min chase were mock-treated or treated with TPCK-trypsin (20 #g) for 30 min at 37°C. Samples were diluted tenfold in MNT, and aliquots were loaded on sucrose gradients and centrifuged as described in Materials and Methods. Gradient fractions were collected, immunoprecipitated, and analyzed by SDS-PAGE. The trypsin-treated samples are shown in Fig. 9 . Trypsin digestion did not cause dissociation of VSV G trimers (compare Fig. 9 A with Fig. 5) . Significantly, the larger proteolytic product of Gml migrated at the bottom of the gradient, whereas some of the smaller product pelleted and some was found throughout the gradient (Fig. 9 B) . This suggested that removing part of the cytoplasmic tail of Gml does not disrupt the oligomer, and further supports the possibility that digestion of SDS-resistant material gives rise to the larger band.