Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4

Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.