AtMAP65-1 binds to tubulin dimers to promote tubulin assembly.
AtMAP65-1 binds to tubulin dimers to promote tubulin assembly.
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DOI：
10.5483/bmbrep.2007.40.2.218
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发表时间：
2007-03
期刊：
影响因子：
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通讯作者：
Hua Li;M. Yuan;Tonglin Mao
中科院分区：
文献类型：
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作者：
Hua Li;M. Yuan;Tonglin Mao
在拟南芥中，微管相关蛋白AtMAP 65 -1在微管动力学和组织中显示出多种功能。然而，AtMAP 65 -1是否与微管蛋白二聚体结合以及它如何调节微管动力学仍然是一个悬而未决的问题。在本研究中，研究了AtMAP 65 -1的微管蛋白结合活性。下拉和共沉降实验表明，AtMAP 65 -1结合到微管蛋白二聚体，在1：1的摩尔比。交联实验表明AtMAP 65 -1通过与微管蛋白异源二聚体的α-微管蛋白相互作用而与微管蛋白二聚体结合。实验结果表明，AtMAP 65 - 1通过与微管蛋白二聚体相互作用促进微管蛋白的组装。此外，AtMAP 65 -1的五个截短版本，即AtMAP 65 -1 deltaN 339（氨基酸340-587）; AtMAP 65 -1 deltaN 494（氨基酸495-587）; AtMAP 65 -1 340-494（氨基酸340-494）; AtMAP 65 -1 deltaC 495（氨基酸1-494）和AtMAP 65 -1 deltaC 340（氨基酸1-339）的结合活性和在体外微管蛋白聚合中的作用。5个截短蛋白中有4个（AtMAP 65 -1 deltaN 339、deltaN 494、AtMAP 65 -1 340-494和deltaC 495）能够与微管共沉淀，其中3个（AtMAP 65 -1 deltaN 339、deltaN 494和AtMAP 65 -1 340-494）能够与微管蛋白二聚体结合。在三种截短蛋白中，AtMAP 65 -1 deltaN 339显示出最大的促进微管蛋白聚合的活性，AtMAP 65 -1 deltaN 494显示出与全长蛋白几乎相同的促进微管蛋白组装的活性，而AtMAP 65 -1 340-494具有较小的促进微管蛋白组装的活性。与此相反，AtMAP 65 -1 deltaC 495，结合微管，但不微管蛋白二聚体，不影响微管蛋白的组装。我们的研究表明AtMAP 65 -1可能通过与微管蛋白二聚体结合来促进微管蛋白的组装。
In Arabidopsis thaliana, the microtubule-associated protein AtMAP65-1 shows various functions on microtubule dynamics and organizations. However, it is still an open question about whether AtMAP65-1 binds to tubulin dimers and how it regulates microtubule dynamics. In present study, the tubulin-binding activity of AtMAP65-1 was investigated. Pull-down and co-sedimentation experiments demonstrated that AtMAP65-1 bound to tubulin dimers, at a molar ratio of 1 : 1. Cross-linking experiments showed that AtMAP65-1 bound to tubulin dimers by interacting with alpha-tubulin of the tubulin heterodimer. Interfering the bundling effect of AtMAP65-1 by addition of salt and monitoring the tubulin assembly, the experiment results indicated that AtMAP65-1 promoted tubulin assembly by interacting with tubulin dimers. In addition, five truncated versions of AtMAP65-1, namely AtMAP65-1 deltaN339 (amino acids 340-587); AtMAP65-1 deltaN494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 deltaC495 (amino acids 1-494) and AtMAP65-1 deltaC340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 deltaN339, deltaN494, AtMAP65-1 340-494 and deltaC495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 deltaN339, deltaN494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 deltaN339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 deltaN494 exhibited almost the same activity as the full length protein in promoting tubulin assembly, and AtMAP65-1 340-494 had minor activity to promote tubulin assembly. On the contrast, AtMAP65-1 deltaC495, which bound to microtubules but not to tubulin dimers, did not affect tubulin assembly. Our study suggested that AtMAP65-1 might promote tubulin assembly by binding to tubulin dimers in vivo.