Identification of novel peptide binding proteins in the endoplasmic reticulum: ERp72, calnexin, and grp170.

Transient interactions between molecular chaperones and nascent polypeptide chains assist protein folding in the endoplasmic reticulum. In an experimental setting that resembles the ER, we have used peptides as model substrates to identify and compare substrate specificities of ER-resident chaperones. The ER-located peptide transporter TAP was used to introduce peptides into the lumen of microsomes. In addition to PDI and gp96, previously identified as peptide-binding chaperones in the ER, we show that ERp72, calnexin, and grp170 interact with TAP-translocated peptides. The chaperones that have been identified can all bind peptide substrates that range from 8 to 40 amino acids in a manner independent of ATP. In addition, these chaperones exhibit broad and largely overlapping, however not identical, substrate selectivities. Our data indicate that peptide translocation into microsomes via TAP can be used as a method to monitor substrate selectivities of ER-resident chaperones. The implications of the observed preferences for chaperone-substrate interactions and for chaperones applied as vehicles in peptide-based vaccination strategies will be discussed.