Background: Newly synthesised peptide-receptive major histocompatibility complex (MHC) class I molecules form a transient loading complex in the endoplasmic reticulum with the transporter associated with antigen processing (TAP) and a set of accessory proteins. Binding of peptide to the MHC class I molecule is necessary for dissociation of the MHC class I molecule from the complex with TAP, but other components of the complex might also be involved. To investigate the role of TAP in this process, mutations that block nucleotide binding were introduced into the ATP-binding site of TAP.
Results: Mutant TAP formed apparently normal loading complexes with MHC class I molecules and accessory components, but had no nucleotide-binding or peptide-transport activity. Nevertheless, whereas wild-type loading complexes in detergent lysates could be dissociated by addition of peptides that bind MHC class I molecules, mutant complexes could not be dissociated in this way. Depletion of nucleotide diphosphates or triphosphates from wild-type lysates blocked peptide-mediated dissociation of MHC class I molecules, which could be reversed by readdition of nucleotide diphosphates or triphosphates. Complexes between mutant TAP and MHC class I molecules remained associated in vivo until they were degraded. Disruption of nucleotide binding also eliminated TAP's peptide-binding activity.
Conclusions: Peptide-mediated dissociation of the MHC class I molecule from the loading complex depends on conformational signals arising from TAP. Integrity of the nucleotide-binding site is required not only for transmission of this conformational signal to the loading complex, but also for binding of peptide to TAP. Thus, the dynamic activity of the loading complex is synchronised with the nucleotide-mediated peptide-binding and transport cycle of TAP.