TAP ASSOCIATES WITH A UNIQUE CLASS-I CONFORMATION, WHEREAS CALNEXIN ASSOCIATES WITH MULTIPLE CLASS-I FORMS IN MOUSE AND MAN

To define the rules governing de novo assembly of the trimeric class I complex, we have identified the class I folding/assembly intermediate s associated with calnexin or TAP, using both human and mouse cell lin es. To better characterize the class I H chain structure associated wi th TAP, mouse mAb that distinguish open (64-3-7(+)) vs folded (30-5-7( +)) L(d) heavy (H) chains were used, We report here that open forms of L(d) are uniquely and specifically associated with TAP and that the c onformational change in the class I H chain coincident with peptide bi nding induces TAP release, Chimeric L(d)/Q10 displayed TAP association , demonstrating that soluble class I molecules can bind TAP, As previo usly reported, beta(2)m was found to be required for H chain associati on with TAP. Interestingly, beta(2)m was associated with TAP in the hu man class I-negative cell line LCL 721.221, suggesting that beta(2)m c an bind to TAP before class I H chain, In contrast to TAP, which binds a specific class I conformation, calnexin was detected in association with multiple forms of both mouse and human class I. Most significant ly, we show for the first time that beta(2)m-assembled forms of human as well as mouse class I molecules interact with calnexin. Based on th ese findings, we propose a model for the sequential assembly of class I heterotrimers and their respective interactions with TAP and calnexi n.