Characterization of the motility of monomeric kinesin-5/Cin8
Characterization of the motility of monomeric kinesin-5/Cin8
复制标题
单体驱动蛋白-5/Cin8 运动性的表征
DOI：
10.1016/j.bbrc.2021.03.134
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发表时间：
2021
影响因子：
3.1
通讯作者：
Yajima Junichiro
中科院分区：
文献类型：
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作者：
Yamagishi Masahiko;Maruyama Yohei;Sugawa Mitsuhiro;Yajima Junichiro
Cin 8是酿酒酵母（Saccharomycesaekinesin-5）的一种蛋白质，在有丝分裂中起重要作用。Inin vitromotility测定，四聚体和二聚体Cin 8构建体显示响应于离子强度或Cin 8运动密度的双向运动。然而，属性转换方向性是否存在于单体形式的Cin 8是未知的。在这里，我们设计了在环8结构域中具有和不具有Cin 8特异性CIN 99残基的单体Cin 8构建体，并在马达密度或离子强度的范围内使用体外极性标记的微管滑动测定来检查这些构建体的方向性。我们发现，两种单体构建体在测量的范围内仅显示正末端定向活性，这表明由Cin 8驱动的负末端定向运动性对于至少二聚体形式是必要的。使用一个在体外微管螺旋实验，我们还发现，单体Cin 8螺旋微管围绕其纵轴与一个恒定的左手间距。总的来说，我们的研究结果意味着，加端定向和左手运动活动包括与其他单体N-驱动蛋白的Cin 8电机域的固有特性。
Cin8, theSaccharomyces cerevisiaekinesin-5, has an essential role in mitosis. Inin vitromotility assays, tetrameric and dimeric Cin8 constructs showed bidirectional motility in response to ionic strength or Cin8 motor density. However, whether property-switching directionality is present in a monomeric form of Cin8 is unknown. Here we engineered monomeric Cin8 constructs with and without the Cin8-specific ∼99 residues in the loop 8 domain and examined the directionality of these constructs using anin vitropolarity-marked microtubule gliding assay within the range of the motor density or ionic strength. We found that both monomeric constructs showed only plus end-directed activity over the ranges measured, which suggested that minus end-directed motility driven by Cin8 is necessary for at least dimeric forms. Using anin vitromicrotubule corkscrewing assay, we also found that monomeric Cin8 corkscrewed microtubules around their longitudinal axes with a constant left-handed pitch. Overall, our results imply that plus-end-directed and left-handed motor activity comprise the intrinsic properties of the Cin8 motor domain as with other monomeric N-kinesins.