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

To ask if Gml oligomerization was an artifact generated during SDS-PAGE, we analyzed oligomer formation using velocity gradient sedimentation in sucrose (Doms et al., 198% Gml does not occur at 16°C. Transfected HeLa cells were metabolically radiolabeled for 5 min, and then chased at 16 or 37°C for up to 90 rain. One set of dishes was incubated for 90 min at 16°C, then transferred to 37°C for 60 min. VSV G and related proteins were immunoprecipitated from solubilized cells as described in Materials and Methods. 1988 ). HeLa cells were transiently transfected with VSV G or Gml, and then metabolically radiolabeled for 5 min and solubilized either immediately or after 60 min of chase. Lysates were loaded onto 5-20% sucrose gradients, centrifuged, and analyzed as described in Materials and Methods. Immediately after synthesis, VSV G migrated as a 4S20.~ peak (Fig. 5; Doms et al., 1987) . After a 60-min chase, all of the VSV G had trimerized, migrating as a discrete 8S20,~ peak. The behavior of Gml in these gradients was markedly different. Immediately after synthesis, Gml migrated similarly to the VSV G monomer. After a 60-rain chase, however, much of the Gml was found in the pellet in an SDS-resistant form ( Fig. 5 ; note top of lane). The timedependent shift in the mobility of the oligomer in sucrose gradients suggests that oligomers already exist in the cells at the time of lysis and immunoprecipitation. The SDSsensitive material in the Gml pellet fractions may represent an intermediate form of oligomer that is not yet SDSresistant. Alternatively, this material could contain protein released from the SDS-resistant oligomer during SDS-PAGE. In contrast to our findings with Gml, misfolded mutants of VSV G that aggregate in the ER are found in the pellet (and are SDS-soluble) immediately after synthesis (Doms et al., 1988) . Using different centrifugation conditions, we estimated the average size of the Gml oligomer to be approximately 22S20.~, or roughly 800 kD (data not shown). Therefore, if Gml (MW 66 kD) is the sole component of the oligomer, we estimate there would be ,o12 molecules per oligomer.