Keusekotten Kirstin, Leonhardt Ralf M, Ehses Sarah, Knittler Michael R
Institute for Genetics, University of Cologne, Zuelpicher Strasse 47, D-50674 Cologne, Germany.
J Biol Chem. 2006 Jun 30;281(26):17545-51. doi: 10.1074/jbc.M602360200. Epub 2006 Apr 19.
The transporter associated with antigen processing (TAP) is essential for the delivery of antigenic peptides from the cytosol into the endoplasmic reticulum (ER), where they are loaded onto major histocompatibility complex class I molecules. TAP is a heterodimeric transmembrane protein that comprises the homologous subunits TAP1 and TAP2. As for many other oligomeric protein complexes, which are synthesized in the ER, the process of subunit assembly is essential for TAP to attain a native functional state. Here, we have analyzed the individual requirements of TAP1 and TAP2 for the formation of a functional TAP complex. Unlike TAP1, TAP2 is very unstable when expressed in isolation. We show that heterodimerization of TAP subunits is required for maintaining a stable level of TAP2. By using an in vitro expression system we demonstrate that the biogenesis of functional TAP depends on the assembly of preexisting TAP1 with newly synthesized TAP2, but not vice versa. The pore forming core transmembrane domain (core TMD) of in vitro expressed TAP2 is necessary and sufficient to allow functional complex formation with pre-existing TAP1. We propose that the observed assembly mechanism of TAP protects newly synthesized TAP2 from rapid degradation and controls the number of transport active transporter molecules. Our findings open up new possibilities to investigate functional and structural properties of TAP and provide a powerful model system to address the biosynthetic assembly of oligomeric transmembrane proteins in the ER.
与抗原加工相关的转运体（TAP）对于将抗原肽从胞质溶胶转运至内质网（ER）至关重要，抗原肽在内质网中被加载到主要组织相容性复合体I类分子上。TAP是一种异源二聚体跨膜蛋白，由同源亚基TAP1和TAP2组成。与许多在内质网中合成的其他寡聚蛋白复合物一样，亚基组装过程对于TAP达到天然功能状态至关重要。在此，我们分析了TAP1和TAP2形成功能性TAP复合物的各自需求。与TAP1不同，单独表达时TAP2非常不稳定。我们发现TAP亚基的异源二聚化对于维持TAP2的稳定水平是必需的。通过使用体外表达系统，我们证明功能性TAP的生物发生取决于预先存在的TAP1与新合成的TAP2的组装，反之则不然。体外表达的TAP2的孔形成核心跨膜结构域（核心TMD）对于与预先存在的TAP1形成功能性复合物是必要且充分的。我们提出观察到的TAP组装机制可保护新合成的TAP2免于快速降解，并控制运输活性转运体分子的数量。我们的发现为研究TAP的功能和结构特性开辟了新的可能性，并提供了一个强大的模型系统来研究内质网中寡聚跨膜蛋白的生物合成组装。
