Functional non-equivalence of ATP-binding cassette signature motifs in the transporter associated with antigen processing (TAP)
Functional non-equivalence of ATP-binding cassette signature motifs in the transporter associated with antigen processing (TAP)
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DOI：
10.1074/jbc.m404042200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Tampé, R
中科院分区：
文献类型：
--
作者：
Chen, M;Abele, R;Tampé, R
与抗原加工相关的转运蛋白（TAP）是细胞免疫系统的关键组成部分。作为 ATP 结合盒 (ABC) 超家族的成员，TAP 水解 ATP，为肽从细胞质转运到内质网腔提供能量。 TAP 由 TAP1 和 TAP2 组成，各自包含一个跨膜结构域和一个核苷酸结合结构域 (NBD)。在这里，我们研究了 ABC 签名基序（C 环）对 NBD 功能非等价性的作用，其中包含 TAP1 的规范 C 环（LSGGQ）和 TAP2 的简并 C 环（LAAGQ）。 TAP1 中亮氨酸或甘氨酸 (LSGGQ) 的突变完全废除了肽转运。然而，具有 TAP2 等效突变的 TAP 复合物仍然显示出残留的肽转运活性。为了阐明 TAP NBD 不对称的起源，我们进一步检查了具有交换 C 环的 TAP 复合物。引人注目的是，具有两个规范 C 环的嵌合体显示出最高的转运速率，而具有两个简并 C 环的嵌合体具有最低的转运速率，这表明 ABC 特征基序控制肽转运效率。所有单位点突变体和嵌合体在肽或 ATP 结合方面表现出相似的活性，这意味着这些突变影响 TAP 的 ATP 酶活性。此外，这些结果证明，C 环的丝氨酸对于 TAP 功能不是必需的，而是与 C 环的其他残基一起协调两个核苷酸结合位点的 ATP 水解。
The transporter associated with antigen processing ( TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine ( LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.