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

To demonstrate the location of the fragment C within the polypeptide chain directly, we introduced a gradient of label into Ii chain, mRNA from spleen cells was translated in the presence of microsomal membranes. 1 min after the start of translation, 7-methyl-guanosine was added to synchronize mRNA translation. After different time intervals (Fig. 3) , unlabeled methionine was added to a final concentration of 5-7) . Ii antigens were immunoprecipitated and incubated with no proteas¢ (lanes 1, 4, and 5) or with 5 t~g/ml (lanes 2 and 6) or 50 ~g/ml V8 protease (lanes 3 and 7) for 15 rain at 30"C. The positions ofglycosylated Ii chain, the 41-kD polypeptide, and fragments A, B, and C which are generated upon cleavage with Staphylococcus aureus V8 protease are indicated, protease, and characterized by SDS PAGE and autoradiography. The amount of label in fragments A, B, and C was then determined by densitometry of the autoradiograph (Fig. 3) . It can be predicted that label in a polypeptide fragment located close to the NH2-terminal end will remain constant, whereas label in fragments located close to the COOH-terminal end will increase with time of the pulse. As can be seen in Fig. 3 , the label in fragment C remains constant while label in fragments A and B increases with the length of the pulse. This clearly demonstrates that fragment C is located close to the NH2-terminal end of Ii chain. To demonstrate that the C fragment from in vivo synthesized Ii chain is also recognized by In-1 antibody, we characterized Ii chain and its fragments by immunoblotting with In-1 antibody. Total protein from CH 1.1. cells either before or after digestion with Staphylococcus aureus V8 protease was separated by SDS PAGE, blotted onto nitrocellulose filter, and developed with In-I antibody. Fig. 4 shows that Ii chain and the 41-kD protein are recognized by In-1 antibody (lane 1) as well as after V8 protease digestion the 10-kD fragment C (lane 2). Thus, we conclude that also in vivo the NH2terminal fragment ofli chain carries the determinant required for the binding to In-1 antibody.