Peptide-bound major histocompatibility complex class I molecules associatewith tapasin before dissociation from transporter associated with antigen processing

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then tr anslocated across the membrane of the endoplasmic reticulum by the transpor ter associated with antigen processing (TAP). TAP is a trimeric complex con sisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti TAP1/2 and anti-tapasin antibodies, re spectively. TAP1 and TAP2 are required for the peptide transport. Tapasin i s involved in the association of class I with TAP and in the assembly of cl ass I with peptide. The mechanisms of tapasin function are still unknown. M oreover, there has been no evidence for a murine tapasin analogue, which ha s led to the suggestion that murine MHC class I binds directly to TAP1/2. I n this study, we have cloned the mouse analogue of tapasin. The predicted a mino acid sequence showed 78% identity to human tapasin with identical cons ensus sequences of signal peptide, N-linked glycosylation site, transmembra ne domain and double lysine motif. However, there was less homology (47%) f ound at the predicted cytosolic domain, and in addition, mouse tapasin is 1 4 amino acids longer than the human analogue at the C terminus. This part o f the molecule may determine the species specificity for interaction with M HC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/ 2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I w ere coprecipitated by anti-tapasin antiserum indicative of association of t apasin with TAP1 but not TAP2. With crosslinker-modified peptides and purif ied microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules i n the endoplasmic reticulum.