EVIDENCE FOR SUCCESSIVE PEPTIDE BINDING AND QUALITY-CONTROL STAGES DURING MHC CLASS-I ASSEMBLY

Intracellular antigens are continually presented to cytotoxic T lympho cytes by major histocompatibility complex (MHC) class I molecules, whi ch consist of a polymorphic 43 kDa heavy chain and a 12 kDa soluble su bunit beta(2)-microglobulin (beta(2)m), and which bind an 8-10 amino-a cid antigenic peptide, The assembly of this trimolecular complex takes place in the lumen of the endoplasmic reticulum (ER) [1] and almost c ertainly requires cofactors, Most MHC class I molecules in the ER that have not yet acquired peptide are simultaneously bound to the transpo rter associated with antigen processing (TAP), to the 48 kDa glycoprot ein tapasin and to the lectin-like chaperone calreticulin, in a multic omponent 'loading complex' [2], Previous studies have shown that a mut ant MHC class I molecule T134K (in which Thr134 was changed to Lys) fa ils to bind to TAP [3], Here, we show that this point mutation also di srupted, directly or indirectly, the interaction between MHC class I m olecules and calreticulin. T134K molecules did not present viral antig ens to T cells even though they bound peptide and beta(2)m normally in vitro. They exited the ER rapidly as 'empty' MHC class I complexes, u nlike empty wild-type molecules which are retained in the ER and degra ded. We show here that, paradoxically, the rapid exit of empty T134K m olecules from the ER was dependent on a TAP-derived supply of peptides . This implies that MHC class I assembly is a two-stage process: initi al binding of suboptimal peptides is followed by peptide optimisation that depends on temporary ER retention.