Invariant (Ii) chain is a membrane-spanning protein that is found associated intracellularly with class II histocompatibility antigens. In the endoplasmic reticulum Ii chain spans the membrane and exposes the NH2 terminus on the cytoplasmic and the COOH terminus on the lumenal side. This orientation across the membrane is demonstrated directly with the monoclonal antibody In-l, which exclusively recognizes the NH2 terminal cytoplasmically exposed part of Ii chain. Membrane insertion of Ii chain requires signal recognition particle and docking protein. When tested in a wheat germ cell free system, signal recognition particle arrests translation of Ii chain. No signal sequence is cleaved from Ii chain upon membrane insertion.
The signal recognition particle (SRP) has been shown to target nascent secretory and membrane proteins to the endoplasmic reticulum. In the wheat germ cell-free system, SRP arrests the elongation of the nascent chains until the translational complex is docked to the endoplasmic reticulum membrane where the interaction between SRP and docking protein causes a release of the nascent chain arrest. For two secretory proteins, arrested peptides of 70 amino acids have been identified (Walter, P., Ibrahimi, I., and Blobel, G. (1981) J. Cell Biol. 91, 545-550; Meyer, D. I., Krause, E., and Dobberstein, B. (1982) Nature 297, 647-650). By using an in vitro coupled transcriptiontranslation system, we have analyzed SRP arrest and the resulting peptides of the two secretory proteins lysozyme and granulocyte-macrophage colony-stimulating factor and the membrane protein invariant chain. SRP arrested the elongation of all three proteins at multiple sites, giving rise to ladders of arrested peptides. The size of the arrested peptides increased with the time of translation, resulting in mostly fulllength pre-peptides after about 40 min. This suggests that SRP arrest is transient rather than stable. Upon addition of microsomes, the SRP arrest was released, and all the blocked peptides could be chased into maturf proteins or full-length precursors.
The invariant (Ii) chain is a membrane-spanning glycoprotein found intracellularly associated with class II major histocompatibility complex (MHC) molecules. Using hybridselected translation and the Ii-specific monoclonal antibody In-1, we have isolated a cDNA clone (pli-5) coding for most of the Ii chain. Sequence analysis of this clone reveals an open reading frame encoding 169 amino acid residues. The protein is rich in methionine and contains two potential N-glycosylation sites. No stretch of uncharged amino acid residues, characteristic for a membrane-spanning segment, is found close to the COOH-terminal end. There is one, however, close to the NH2-terminal end. As it is known that -20 amino acid residues of Ii chain are exposed on the cytoplasmic side, we conclude that the Ii chain spans the membrane exposing the NH2 terminus on the cytoplasmic side and the COOH terminus on the luminal side.
We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.