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

To characterize the disposition of Ii chain across the membrane of the ER, we translated mRNA from mouse spleen cells in a wheat germ cell free system in the presence of microsomal membranes derived from dog pancreas. In such a system membrane proteins become inserted into the membrane asymmetrically in the same way as in the cell and the orientation across the membrane can be determined by protease treatment (24, 37) . Protease will cleave only that part of a membrane protein that is exposed on the cytoplasmic side, whereas the lumenally disposed part is protected by the membrane. The orientation of the protein across the membrane can then be deduced, if the means exist for detecting the COOH or NH2 termini of the protein. Since we could make use of a monoclonal antibody specific for Ii chain, we tested whether the antibody recognizes exclusively either the lumenally or the cytoplasmically disposed portion of Ii chain. Depending on the location of the antigenic determinant of In-1 we would be able to discriminate between the two possible orientations of Ii chain across the membrane.