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

In vitro synthesized proteins were digested with protease by adding 1 vol of 2% Triton X-100, 100 mM Tris HCI, oH 7.5, 300 mM NaCI, and Staphylococcus aureus V8 protease to a final concentration of 50 #g/ml and incubating the sample for 15 rain at 30"C. Proteolysis was stopped by addition of phenylmethylsulfonyl fluoride (20 #g/ml). For proteolysis of immunoadsorbed li chain, In-1 antibody and protein A-Sepharose were added to either a CH 1.1. cell lysate or a cell free lysate (12) . The mixture was incubated for 60 rain and the Sepharose beads with bound IgG and Ii chains washed and resuspended in 20 #1 of l0 mM Tris-Ha pH 7.5 and 50 ~g/ml of Staphylococcus aureus V8 protease. After incubation for 15 rain at 30"C, protein fragments were immediately analyzed by SDS PAGE.