Structural insight into the membrane insertion of tail-anchored proteins by Get3
Structural insight into the membrane insertion of tail-anchored proteins by Get3
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DOI：
10.1111/j.1365-2443.2009.01362.x
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发表时间：
2010-01-01
期刊：
影响因子：
2.1
通讯作者：
Fukai, Shuya
中科院分区：
文献类型：
--
作者：
Yamagata, Atsushi;Mimura, Hisatoshi;Fukai, Shuya
尾部锚定(Tail Anchored，TA)蛋白是由C末端附近的一个跨膜区(TMD)定义的，在许多细胞过程中都是重要的。TA蛋白的膜插入是由高度保守的ATPase Get3介导的。在这里，我们报道了Get3在3.0A和2.8A分辨率下分别以ADP结合和无核苷酸形式的晶体结构。Get3由一个核苷酸结合区和一个螺旋结构域组成。这两种结构都显示出由锌离子介导的头对头取向的同源二聚体，代表开放的二聚体构象。我们的交联实验表明，闭合二聚体刺激了ATP的水解，这可能与TA-蛋白的释放有关。此外，我们使用一个模型TA蛋白Sec22p进行的基于共表达的结合分析表明，Get3的螺旋结构域与Sec22p TMD之间存在直接的相互作用。这种相互作用与三磷酸腺苷和二聚体的形成无关。最后，我们提出了一种结构机制，将ATP水解与保守的DTAPTGH基序介导的TA蛋白插入联系起来。
Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C-terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP-bound and nucleotide-free forms at 3.0 A and 2.8 A resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn2+-mediated homodimer in a head-to-head orientation, representing an open dimer conformation. Our cross-link experiments indicated the closed dimer-stimulating ATP hydrolysis, which might be coupled with TA-protein release. Further, our coexpression-based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA-protein insertion mediated by the conserved DTAPTGH motif.