ER aminopeptidases generate a unique pool of peptides for MHC class I molecules

We define here the specificity and significance of proteases in the endoplasmic reticulum (ER) that generate peptides for presentation by major histocompatibility complex (MHC) class molecules. We show that aminopeptidases efficiently trimmed all residues except proline that flank the NH2-termini of antigenic precursors in the ER and caused an accumulation of X-P-X-n peptides. An aminopeptidase inhibitor blocked peptide trimming in the ER and, consequently, the generation of peptide-loaded MHC molecules. Peptide trimming in the ER is therefore a key step in the MHC class I antigen-processing pathway and also explains the paradox of why many MHC class molecules display peptides with the X-P-X. motif despite the inability of the transporter associated with antigen processing to transport such peptides from the cytoplasm.