Knittler M R, Alberts P, Deverson E V, Howard J C
Institute for Genetics University of Cologne D-50674, Cologne, Germany.
Curr Biol. 1999 Sep 23;9(18):999-1008. doi: 10.1016/s0960-9822(99)80448-5.
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.
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.
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.
新合成的肽感受性主要组织相容性复合体（MHC）I类分子在内质网中与抗原加工相关转运体（TAP）及一组辅助蛋白形成一个短暂的装载复合体。肽与MHC I类分子的结合对于MHC I类分子从与TAP的复合体中解离是必要的，但该复合体的其他组分可能也参与其中。为了研究TAP在此过程中的作用，将阻断核苷酸结合的突变引入TAP的ATP结合位点。
突变型TAP与MHC I类分子及辅助组分形成了明显正常的装载复合体，但没有核苷酸结合或肽转运活性。然而，虽然去污剂裂解物中的野生型装载复合体可通过添加与MHC I类分子结合的肽而解离，但突变复合体却不能以这种方式解离。从野生型裂解物中去除二磷酸核苷酸或三磷酸核苷酸可阻断肽介导的MHC I类分子的解离，重新添加二磷酸核苷酸或三磷酸核苷酸可使其逆转。突变型TAP与MHC I类分子之间的复合体在体内一直保持结合状态直至被降解。核苷酸结合的破坏也消除了TAP的肽结合活性。
肽介导的MHC I类分子从装载复合体中的解离取决于TAP产生的构象信号。核苷酸结合位点的完整性不仅是将此构象信号传递至装载复合体所必需的，也是肽与TAP结合所必需的。因此，装载复合体的动态活性与TAP的核苷酸介导的肽结合及转运循环是同步的。
