In the endoplasmic reticulum, MHC class II ab dimers associate with the trimeric invariant chain (li), generating a nine-subunit(abli)3 complex. In the presence of li, the peptide binding groove is blocked, so that loading with self or antigenic peptides can only occur after proteolytic removal of li in specialized post-Golgi compartments. The class 11-associated invariant chain peptide region of li (about residues 81-1 04) is known to mediate binding to class II molecules and blockade of the groove, but this does not exclude additional contact sites for li. Using a set of overlapping li peptides and recombinant soluble li, we demonstrate here that a large segment of Ii encompassing approximately residues 71 to 128 interacts with HLA-DR molecules. The N- and C-terminal regions of this Ii segment appear to bind outside the peptide groove to the contact area for the staphylococcal superantigen Staphylococcus aureus enterotoxin B on the a1 domain. The core region of this segment (residues 95-108)prevents binding of antigenic peptides, probably by interaction with the peptide groove. Occupation of the groove with antigenic peptides abolishes binding not only of the core region, but also that of those Ii peptides that bind outside the groove. These findings suggest the existence of distinct conformational states of class II molecules, with Ii binding preferentially to one form.
Translocation of proteins across the endoplasmic reticulum membrane is initiated by the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein complex consisting of a 7S RNA and six polypeptides. To investigate the functions of the SRP components, we have tested the activities of several SRP subparticles. We show that the SRP GTPase (SRP54) alone binds a signal sequence and discriminates it from a non-signal sequence. Although SRP54 alone is unable to promote translocation, SRP54 in a complex with SRP RNA is both necessary and sufficient to promote translocation of an elongation-arrested nascent protein in a GTPregulated manner. For co-translational translocation, additional SRP components are required. We discuss the implications of our results for the function of the Escherichia coli SRP which is homologous to the SRP54/SRP-RNA complex.
Lipids and proteins were found to contact a nascent type II membrane protein, as well as a nascent secretory protein, during their insertion into the membrane of the endoplasmic reticulum. This suggests that the protein-conducting channel is open laterally toward the lipid bilayer during an early stage of protein insertion. Contact to lipids was confined to the hydrophobic core region of the respective signal or signal anchor sequence. Thus, the nascent polypeptide is positioned in the translocation complex such that the signal or signal anchor sequence faces the lipid bilayer, whereas the hydrophilic, translocating portion is in proteinaceous environment.