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KMID : 0380220070400020218
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Journal of Biochemistry and Molecular Biology
2007 Volume.40 No. 2 p.218 ~ p.225
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AtMAP65-1 Binds to Tubulin Dimers to Promote Tubulin Assembly
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Li Hua
Yuan Ming
Mao Tonglin
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Abstract
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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 α-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 &dgr;N339 (amino acids 340-587); AtMAP65-1 &dgr;N494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 &dgr;C495 (amino acids 1-494) and AtMAP65-1 &dgr;C340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 &dgr;N339, &dgr;N494, AtMAP65-1 340-494 and &dgr;C495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 &dgr;N339, &dgr;N494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 &dgr;N339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 &dgr;N494 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 &dgr;C495, 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.
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KEYWORD
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AtMAP65-1, Arabidopsis, Dynamics, Microtubules, Tubulin
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