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

Binding of Proteins to Con A-Sepharose. After translation of spleen cell mRNA in a 25-~1 wheat germ cell free system in the presence of rough microsomes, membranes were pelleted by centrifugation for 20 rain at 10,000 rpm at 4"C, They were then solubilized in 0.5% Triton X-100, 150 mM NaCI, 50 mM Tris-HCl, pH 7.5, 1 mM CaCI2, 1 mM MnCI2, and 4 ug/ml of phenylmethylsulfonyl fluoride and then 50 #1 of Con A-Sepharose was added (a slurry containing beads to solubilization buffer in a 1 : 1 ratio). The mixture was incubated for 60 min at room temperature. Beads were then washed three times with 100 mM acetate at pH 6, 150 mM NaCI, 1 raM MnC1, 1 mM CaCI2, and 1 mM MgCI2. To the pelleted beads sample buffer for SDS PAGE was added and proteins analyzed as described (28) .