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

In contrast to the chimeric Gml protein, oligomers of the IBV M glycoprotein are not detected when assayed by sucrose gradient sedimentation (data not shown). This might suggest that oligomerization is not a retention mechanism for native M protein; alternatively, M oligomers might readily dissociate in detergent. The resistance of the Gml oligomer to solubilization suggests a structural feature of VSV G may stabilize the complex. We asked whether the lumenal domain of VSV G, which is sufficient for trimerization (Doms et al., 1988) , was required for oligomer formation. To do this, we replaced the lumenal domain ofVSV G or Gml with a small soluble glycoprotein, the o~ subunit of hCG. When expressed alone, the oL subunit of hCG is secreted from cells as a monomer (Guan et al., 1988b) . When hCGo~ is fused to the membrane-spanning domain and cytoplasmic tail of VSV G, the chimeric protein (c~m) is membrane-bound and transported to the cell surface (Guan et al., 1988a) . When the VSV G membrane-spanning domain of c~m is replaced with the ml domain of IBV M, the resulting chimera (oemlG) is retained in the Golgi complex (Swift and Machamer, 1991) . Interestingly, in metabolically labeled HeLa cells expressing cxmlG, we observed the time-dependent accumulation of an SDS-resistant species at the interface between the separating and stacking gels (Fig. 6, arrowhead) . This species was not observed in cells expressing am. The smaller l~gure 5. Sucrose gradient sedimentation of Gml and VSV G. HeLa cells transfected with VSV G or Gml were metabolically labeled for 5 rain, and then solubilized immediately or after 60 rain of chase. Lysates were loaded onto 5-20% sucrose gradients and centrifuged as described in Materials and Methods. Fractions were collected, immunoprecipitated using anti-VSV antibody, and analyzed by SDS-PAGE. After the 60-min chase, all of the Gml formed a large oligomer that pelleted under these gradient conditions, and much of it was SDS-resistant. size of the otmlG SDS-resistant oligomer is consistent with the size difference between ctmlG and Gml (28 vs 66 kD). On sucrose gradients, c~mlG solubilized 60 min after synthesis migrated as a larger species (some of which was SDSresistant) than newly synthesized oanlG. In contrast, the mobility of o~m on sucrose gradients was unaltered during the chase (data not shown). Thus the VSV G lumenal domain was not required for SDS-resistant oligomer formation.