The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1 Powerstroke
The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1 Powerstroke
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DOI：
10.1074/jbc.m112.395590
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发表时间：
2012-10-26
影响因子：
4.8
通讯作者：
Gilbert, Susan P.
中科院分区：
文献类型：
--
作者：
Chen, Chun Ju;Porche, Ken;Gilbert, Susan P.
Kar3 是一种酿酒酵母微管负端定向驱动蛋白 14，与 Vik1 或 Cik1 形成二聚体。 Vik1 的 C 端球状结构域呈现驱动蛋白运动结构域的结构，并独立于 Kar3 结合微管，但缺乏核苷酸结合位点。 Kar3Vik1 唯一已知的功能是在有丝分裂期间在纺锤体两极交联平行微管。相比之下，Kar3Cik1 在交配过程中解聚微管，但在有丝分裂过程中交联纺锤体重叠区的反平行微管。最近的一项研究表明，Kar3Vik1 与相邻的微管原丝结合，并使用负端定向动力冲程来驱动 ATP 依赖性运动。这里介绍的前稳态实验扩展了这项研究，并建立了动力冲程机制的 ATP 酶模型。纳入模型的结果表明，Kar3Vik1 通过 Vik1 在 2.4 mu M-1 s(-1) 处与微管碰撞，促进 Kar3 与微管结合，随后在 14 s(-1) 处释放 ADP。 Kar3 与微管的紧密结合破坏了 Vik1 与微管相互作用的稳定性，从而使 Kar3Vik1 处于动力冲程的开始位置。 ATP 与 Kar3 的快速结合与卷曲螺旋茎的旋转相关，并且 26 s(-1) 时的动力冲程后 ATP 水解与 Vik1 无关，这进一步证明 Vik1 在动力冲程期间与卷曲线圈一起旋转。 Kar3Vik1 在 6 s(-1) 时从微管上脱离，完成循环并允许马达返回到其初始构象。结果还揭示了 Kar3Vik1 和 Kar3Cik1 ATP 酶循环的关键差异，支持这两种马达具有独特的生物学功能的事实。
Kar3, a Saccharomyces cerevisiae microtubule minus-end-directed kinesin-14, dimerizes with either Vik1 or Cik1. The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain and binds microtubules independently of Kar3 but lacks a nucleotide binding site. The only known function of Kar3Vik1 is to cross-link parallel microtubules at the spindle poles during mitosis. In contrast, Kar3Cik1 depolymerizes microtubules during mating but cross-links antiparallel microtubules in the spindle overlap zone during mitosis. A recent study showed that Kar3Vik1 binds across adjacent microtubule protofilaments and uses a minus-end-directed power-stroke to drive ATP-dependent motility. The presteady-state experiments presented here extend this study and establish an ATPase model for the power-stroke mechanism. The results incorporated into the model indicate that Kar3Vik1 collides with the microtubule at 2.4 mu M-1 s(-1) through Vik1, promoting microtubule binding by Kar3 followed by ADP release at 14 s(-1). The tight binding of Kar3 to the microtubule destabilizes the Vik1 interaction with the microtubule, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the post-powerstroke ATP hydrolysis at 26 s(-1) is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke. Detachment of Kar3Vik1 from the microtubule at 6 s(-1) completes the cycle and allows the motor to return to its initial conformation. The results also reveal key differences in the ATPase cycles of Kar3Vik1 and Kar3Cik1, supporting the fact that these two motors have distinctive biological functions.