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

Because tGml did not form detectable oligomers, we asked whether preformed Gml oligomers could be disrupted by proteolysis of the cytoplasmic tall. The 29-amino acid Figure 7 . Gml and c~mlG form hetero-oligomers. HeLa cells were transfected with 1 #g each of mlllG and either calf thymus DNA (CT), Gml, VSV G, or Gml~n~. After a 5-min pulse, cells were solubilized either immediately or after a 60-min chase. Aliquots of the solubilized cells were immunoprecipitated with anti-hCG antibody and elcctrophoresed on 12% SDS-polyacrylamide gels. The interface between the stacking and separating gels is marked with an arrowhead, and the mobility of Gml in the gel is denoted with an arrow, amIG forms hetero-oligomers with Gml and Gmlm (but not with VSV G) that could be precipitated by anti-hCG antibody. Fig. 8 A) has numerous arginines and lysines that are susceptible to proteolysis by trypsin. HeLa cells expressing Gml or VSV G were metabolically radiolabeled for 5 min, and then chased for 0 or 60 rain, and microsomes prepared as described in Materials and Methods. Microsomes were treated with TICK-trypsin for 30 rain at 37°C, and then solubilized, immunoprecipitated with anti-VSV antibody, and analyzed on SDS-polyacrylamide gels (Fig. 8 B) . As size standards we used radiolabeled tGml and TMR immunoprecipitated from transfected HeLa cells. Trypsin cleaved newly synthesized VSV G protein to a species migrating slightly larger than TMR on SDS-PAGE (Fig. 8 B, compare lanes 7 and 10) . After 60 rain of chase, two proteolytic products were detected. The upper band corresponded to cleavage of the cytoplasmic tail of mature (sialylated) VSV G, as it was resistant to endoglycosidase H, whereas the lower band was sensitive to endoglycosidase H treatment (not shown). In addition, we frequently observed some residual Gml or VSV G were metabolically labeled for 5 rain, and then chased for 0 or 60 rain. Microst,mes were prepared as described in Materials and Methods and treated with 20 #g TPCK-trypsin (lanes 2, 4, 7, and 9) or mock treated (lanes 1, 3, 6, and 8) for 30 min at 37"C. Proteolysis was stopped by the addition of trypsin inhibitor (20/zg), the microsomes were solubilized, and Gml (lanes 1-4) and VSV G 0anes 6-9) were immunoprecipitated and analyzed by SDS-PAGE. More inside-out microsomes are generated from compartments further along the secretory pathway, accounting for the loss of material recovered after trypsinization of VSV G isolated after a 60-min chase (lane 9). tGml (lane 5) and TMR (lane 10) immunoprecipitated from HeLa cells labeled under the same conditions are included as size standards.