Introduction of a Glycosylation Site into a Secreted Protein Provides Evidence for an Alternative Antigen Processing Pathway: Transport of Precursors of Major Histocompatability Complex Class I–Restricted Peptides from the Endoplasmic Reticulum to the Cytosol

We found that the presentation of a H-2K(d)-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.