AtMAP65-1 binds to tubulin dimers to promote tubulin assembly.
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.
Li
Hua
H
State Key Laboratory of Plant Physiology and Biochemistry; Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
Yuan
Ming
M
Mao
Tonglin
T
D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; C492901:MAP65-1 protein, Arabidopsis; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin