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 intermediates associated with calnexin or TAP, using both human and mouse cell lines. To better characterize the class I H chain structure associated with TAP, mouse mAb that distinguish open (64-3-7+) vs folded (30-5-7+) Ld heavy (H) chains were used. We report here that open forms of Ld are uniquely and specifically associated with TAP and that the conformational change in the class I H chain coincident with peptide binding induces TAP release. Chimeric Ld/Q10 displayed TAP association, demonstrating that soluble class I molecules can bind TAP. As previously reported, beta 2m was found to be required for H chain association with TAP. Interestingly, beta 2m was associated with TAP in the human class I-negative cell line LCL 721.221, suggesting that beta 2m can 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 significantly, we show for the first time that beta 2m-assembled forms of human as well as mouse class I molecules interact with calnexin. Based on these findings, we propose a model for the sequential assembly of class I heterotrimers and their respective interactions with TAP and calnexin.
Carreno
B M
BM
Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Solheim
J C
JC
Harris
M
M
Stroynowski
I
I
Connolly
J M
JM
Hansen
T H
TH
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002461:Cell Line, Transformed; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation
D018528:ATP-Binding Cassette Transporters; D002135:Calcium-Binding Proteins; D015395:Histocompatibility Antigens Class I; D037281:Calnexin