One is enough: functional stoichiometry of tapasin for MHC I antigen presentation

The peptide-loading complex (PLC) is a component of the adaptive immune system, catalyzing the display of the cellular proteome via MHC class I molecules to cytotoxic T-cells. If the presented peptide is derived from a virus or tumor cell, the respective cell is eventually recognized and eliminated by cytotoxic T-cells. The PLC consists of the heterodimeric transporter associated with antigen processing TAP1/2, MHC I, calreticulin, ERp57 and tapasin (Tsn). Tsn connects TAP with MHC I and edits the peptide repertoire on MHC I to ensure the binding of high-affinity peptides. Since the exact composition of the PLC, especially the stoichiometry of Tsn required for MHC I loading, is controversially discussed, we fused Tsn to the N-terminal domains of TAP1 and TAP2, which present the binding site for Tsn. Cells deficient in Tsn showed low MHC I surface expression, while two Tsn molecules symmetrically tethered within the PLC fully restored antigen presentation. Strikingly, only one Tsn molecule linked either to either TAP1 or TAP2 is sufficient for full MHC I loading. Tsn linked to TAP lacking the Tsn binding site, was fully active in peptide loading suggesting that the spacial proximity of TAP and Tsn but not their intermolecular communication are important for efficient loading of MHC I. A dissociation of tapasin from TAP after peptide loading is not required. In addition, co-immunoprecipitation experiments showed that each TAP subunit provides only one binding site for Tsn.