Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.
Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.
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DOI：
10.1083/jcb.200211095
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发表时间：
2003-04-28
影响因子：
7.8
通讯作者：
Mitchison, Tim
中科院分区：
文献类型：
--
作者：
Shirasu-Hiza, Mimi;Coughlin, Peg;Mitchison, Tim
与GMPCPP（一种缓慢水解的GTP类似物）聚合的微管（MT）在缓冲液中稳定，但在爪蟾卵提取物中迅速解聚。这种解聚作用不依赖于三种先前鉴定的MT去稳定剂（Op 18、katanin和XKCM 1/KinI）。我们纯化的因素负责这种新的解聚活性，使用生化分馏和视觉活性测定，并确定为XMAP 215，以前确定为一个突出的MT生长促进蛋白在非洲爪蟾提取物。与纯化结果一致，我们发现XMAP 215是GMPCPP-MT在提取物中去稳定化所必需的，并且重组全长XMAP 215以及NH 2末端片段在体外具有解聚活性。解聚的刺激对MT+末端是特异性的。这些结果提供了一个强大的MT不稳定的活动固有的微管相关蛋白的证据，并表明，不稳定可能是其基本的生化功能的一部分。我们建议，在我们的试验，GMPCPP稳定的MT的基板，作为一个模型的MT末端的暂停状态和XMAP 215的多种活动是统一的拮抗MT暂停的机制。
Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth–promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.