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

When spleen mRNA is translated in the wheat germ cell free system and labeled proteins are immunoprecipitated with monoclonal In-1 antibody, a 25-kD form of Ii chain is selected (Ii') (Fig. !, lane I) . When translation is performed in the presence of microsomal membranes, a 3 l-kD protein is synthesized and this has the same molecular weight as the authentic Ii chain synthesized in vivo (Fig. 1, lane 2) . The latter form is glycosylated, whereas the former is not (see below). Both forms have been described previously (40) . Due to the binding to protein A or Con A, the heavy chains of IgG are also precipitated (Fig. 1, lanes 1, 2, 3, 5, and 6 ). IgG is a typical secretory protein that is translocated across microspinal membranes and therefore can be used to assess the intactness of the microsomal membranes. After digestion with proteinase K no Ii chain-related protein can be precipitated with In-1 antibody (Fig. 1, lane 3) . Heavy chain of IgG, Figure 1 . Protease digestion of Ii chain from intact and detergentsolubilized microsomal vesicles, mRNA from mouse spleen cells was translated in a wheat germ cell free system in the absence (lane 1) or presence of dog pancreas microsomal membranes (lanes 2-7). After translation, proteinase K, 0.5 mg/ml final concentration (lanes 3 and 6), or proteinase K and 0.5% Triton X-100 (lanes 4 and 7) were added and the mixture incubated for 15 min at 30°C. Ii chain was immunoprecipitated using In-i antibody and protein A-Sepharose (lanes 1-4) . Glycoproteins were isolated by binding to Con A-Sepharose (lanes 5-7). Antigens were separated by SDS PAGE and visualized by fluorography. Ii, membrane inserted and glycosylated invariant chain, li', unglycosylated form of Ii chain. H, heavy chain of IgG that binds to protein A-Sepharose and Con A-Sepharose. however, is readily precipitable, demonstrating that a protection against protease digestion has been obtained for proteins located in the lumen of microsomal vesicles. When Triton X-100 is used together with proteinase K, the lumenally located heavy chain of IgG is digested, indicating that protection is dependent on the intact membrane barrier (Fig. l, lanes 4 and 7) .