A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network
A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network
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DOI：
10.1074/jbc.m116.770909
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发表时间：
2017-03-01
影响因子：
4.8
通讯作者：
Kahn, Richard A.
中科院分区：
文献类型：
--
作者：
Francis, Joshua W.;Newman, Laura E.;Kahn, Richard A.
微管动力学涉及微管蛋白二聚体的聚合和解聚，是细胞存活、构筑和分裂所必需的重要和高度调控的过程。微管网络的调节还依赖于α-β-微管蛋白异源二聚体的维持。这些二聚体是复杂的折叠和组装事件的最终结果，需要TCP1环复合体(TRIC或CCT)伴侣和五个微管蛋白特异的伴侣蛋白，微管蛋白结合辅助因子A-E(TBCA-TBCE)。然而，这些伴侣的行为模型是不完整的或不一致的。我们先前从牛组织中纯化了TBCD，并表明它与小GTP酶ARL2紧密结合，但似乎没有活性。在这里，为了确定tbcd的功能形式，并使用非变性凝胶和免疫印迹技术，我们分析了一些小鼠组织和细胞系的裂解产物，以确定tbcd和arl2的四元状态(S)。我们发现这两种蛋白质在天然凝胶中以类似于200 kDa的复合体的形式共同迁移，其中也含有β-微管蛋白。使用人胚胎肾细胞能够纯化细胞和组织裂解物中发现的TBCD中心点ARL2中心点β-微管蛋白三聚体以及另外两个新的TBCD复合体。对破坏与TBCD结合的ARL2点突变的表征表明，ARL2-TBCD相互作用对于细胞中适当维持微管密度至关重要。我们的结论是，TBCD中心点ARL2中心点β-微管蛋白三聚体代表了一个功能复合体，其活性是微管动力学的基础。
Microtubule dynamics involves the polymerization and de-polymerization of tubulin dimers and is an essential and highly regulated process required for cell viability, architecture, and division. The regulation of the microtubule network also depends on the maintenance of a pool of alpha beta-tubulin heterodimers. These dimers are the end result of complex folding and assembly events, requiring the TCP1 Ring Complex (TriC or CCT) chaperonin and five tubulin-specific chaperones, tubulin binding cofactors A-E (TBCA-TBCE). However, models of the actions of these chaperones are incomplete or inconsistent. We previously purified TBCD from bovine tissues and showed that it tightly binds the small GTPase ARL2 but appears to be inactive. Here, in an effort to identify the functional form of TBCD and using non-denaturing gels and immunoblotting, we analyzed lysates from a number of mouse tissues and cell lines to identify the quaternary state(s) of TBCD and ARL2. We found that both proteins co-migrated in native gels in a complex of similar to 200 kDa that also contained beta-tubulin. Using human embryonic kidney cells enabled the purification of the TBCD center dot ARL2 center dot beta-tubulin trimer found in cell and tissue lysates as well as two other novel TBCD complexes. Characterization of ARL2 point mutants that disrupt binding to TBCD suggested that the ARL2-TBCD interaction is critical for proper maintenance of microtubule densities in cells. We conclude that the TBCD center dot ARL2 center dot beta-tubulin trimer represents a functional complex whose activity is fundamental to microtubule dynamics.