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

To locate the first cleavage site in Ii chain, which removes a segment of ~ 1 kD (see Fig. 2, lane 2) , membrane-inserted Ii chain was digested with Staphylococcus aureus V8 protease on intact microsomes and then immunoprecipitated. As no molecular weight shift could be detected under these conditions, it can be concluded that all cleavage sites for V8 protease must be located on the lumenal side (data not shown). In-I antibody recognizes proteolytic fragment C of li chain synthesized by CHI.I. cells. 1 x l 0 7 mouse CHI.1. cells were detergent solubilized and nuclei removed by centrifugation at 5,000 g for 10 min. Solubilized antigens were treated either with no protease (lane 1) or with 5 /~g/ml (lane 2) or 50 #g/ml (lane 3) V8 protease for 15 min at 30"C and then precipitated with 10% TCA, 50% acetone. After resolubilization in SDS-containing sample buffer, proteins were separated by SDS PAGE. After transfer onto nitrocellulose, proteins were reacted with In-I antibody and visualized by peroxidase-coupled anti-rat antibody and diaminobenzidine staining.