Signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane-spanning protein with a cytoplasmically exposed amino terminus

Cell Culture and Labeling of Cells. B-lymphoma cells (CH I. l.) were cultured in RPM1 1640 medium containing 8% fetal calf serum, l0 U penicillin/ streptomycin, 10 #M mereaptocthanol. Cells were washed twice in methioninefree medium and incubated for 20 min at 37"C in the same medium. [3sS] Methionine was added to a final concentration of 400 #Ci/ml, and the cells were incubated at 37*(2 for 60 min. Cells were washed and then solubilized in ice-cold 50 mM Tris-HC1 buffer (pH 7.5) containing 0.15 M NaCl, 5 mM MgCl~, 1% Triton X-100. Protease inhibitor phenylmethylsulfonyl fluoride was added to a final concentration of 20 ~g/ml. Debris were removed by centrifugation at 4"C for 15 rain in a microfuge, and the supernatant was used for immunoprecipitation and SDS PAGE. In some experiments glycosylation of asparagine residues was blocked by adding tunicamycin to the cell culture medium to a final concentration of 3 ug/ml. After incubation for 150 rain at 37°C, cells were labeled as described above.