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

If the oligomerized protein represents a distinct intracellular pool of protein(s), it should not be susceptible to cell surface biotinylation. Moreover, we predicted that an oligomer that formed nonspecifically after cell solubilization would be precipitated by streptavidin since incorporation of a few biotinylated molecules would allow precipitation of the entire oligomer. HeLa cells expressing Gml, VSV G, or Gmlin, were metabolically labeled for 15 min, and then chased for 45 min before cell-surface biotinylation with sulfo-NHSbiotin. After immunoprecipitation, an aliquot of each sample was treated with streptavidin-coupled agarose, and biotinylated and non-biotinylated proteins were recovered and analyzed by SDS-PAGE (Fig. 2) . Under these conditions, ,o50% of the total VSV G was biotinylated and could be recovered in the streptavidin pellet. Only the mature (sialylated) form of VSV G was recovered in this fraction, suggesting that biotinylation was indeed restricted to cell surface proteins. In contrast, essentially none of the Gml was precipitated by streptavidin, suggesting that this protein remained in an intracellular compartment. A small amount of SDS-sensitive Gmli~, was recovered in the streptavidin pellet. More importantly, no SDS-resistant material was biotinylated, suggesting that the SDS-resistant Gmli~ constitutes an intracellular (perhaps Golgi-resident) pool that is distinct from the SDS-sensitive protein, and that only a portion of Gml~, is transported to the plasma membrane where it can be biotinylated. This result strongly suggested that oligomer formation occurs before cell solubilization.