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

For proteins that are translocated across the membrane of the ER, it is known that SRP and DP are required for their insertion into the membrane (33, 42) . For one membrane protein, which is synthesized without cleavable signal sequence, it has been found that SRP and DP are required for membrane insertion but no arrest in elongation was observed (2) . As demonstrated above, Ii chain is synthesized without a cleavable signal sequence, spans the membrane, and exposes the NH2 terminus on the cytoplasmic side. We tested therefore whether this type of protein requires SRP and DP for membrane insertion and whether elongation could be arrested by SRP. When Ii chain was translated in a wheat germ cell free translation system, the 25-kD unglycosylated form ofli chain was synthesized (Fig. 6, lane 1) . When salt-washed rough microsomes, depleted of SRP, were added, no shift of molecular weight could be detected (Fig. 6, lane 2) . When SRP alone was added to the cell free translation system, synthesis of Ii chain was arrested as well as that of heavy chain of IgG (Fig. 6, lane 3) . The arrest in translation could be released when salt-washed rough microsomes (RMk) were added 15 min after initiation of translation (Fig. 6, lane 4) . Glycosylated Ii chains now accumulated. amino acid residues in the Ii chain. The remaining 170 amino acids are protected by the membrane barrier and must thus be located on the lumenal side of the membrane. (c) The monoclonal antibody In-1 recognizes a determinant located on a part of the Ii chain that is exposed on the cytoplasmic side of the membrane. Protease digestion of intact microsomal vesicles destroys this binding site. It is the NH2 terminally located fragment C which is recognized by In-l antibody. Two independent methods were used to locate fragment C within Ii chain: (i) After digestion of Ii chain with Staphylococcus aureus V8 protease, fragment C was found to be the only one that labeled with [3SS]cysteine. The only cysteine in Ii chain occurs 28 amino acid residues away from the NHEterminal end (see Fig. 7 ). (ii) When a gradient of [3SS]cysteine label was introduced into Ii chain, the amount of label in fragment C remained constant throughout the chase period. This is only consistent with a location of fragment C close to NHE-terminal end. (d) There is no cleavable signal sequence found in Ii chain. The unglycosylated precursor of Ii chain synthesized in the wheat germ cell free system in the absence of microsomal membranes has the identical molecular weight as unglycosylated and membrane-inserted Ii chain synthesized by CHI.1 cells. Cleavable signal sequences have a length between 13 and 45 amino acid residues (45) . A difference of five amino acids would have been detected by the SDS PAGE system used.