Abstract
The assembly of major histocompatibility complex (MHC) class I molecules with their peptide ligands in the endoplasmic reticulum (ER) requires the assistance of many proteins that form a multimolecular assemblage termed the 'peptide-loading complex'. Tapasin is the central stabilizer of this complex, which also includes the transporter associated with antigen processing (TAP), MHC class I molecules, the ER chaperone, calreticulin, and the thiol-oxidoreductase ERp57. In the present report, we investigated the requirements of these interactions for tapasin protein stability and MHC class I dissociation from the peptide-loading complex. We established that tapasin is stable in the absence of either TAP or MHC class I interaction. In the absence of TAP, tapasin interaction with MHC class I molecules is long-lived and results in the sequestration of existing tapasin molecules. In contrast, in TAP-sufficient cells, tapasin is re-utilized to interact with and facilitate the assembly of many MHC class I molecules sequentially. Furthermore, chemical cross-linking has been utilized to characterize the interactions within this complex. We demonstrate that tapasin and MHC class I molecules exist in a 1 : 1 complex without evidence of higher-order tapasin multimers. Together these studies shed light on the tapasin protein life cycle and how it functions in MHC class I assembly with peptide for presentation to CD8(+) T cells.