裂变酵母激酶-6 Klp9的两个盘绕结构域是电机四聚化和纺锤体伸长所必需的。

microPublication biology Pub Date : 2025-09-22 eCollection Date: 2025-01-01 DOI:10.17912/micropub.biology.001829
Mason T Nguyen, Liang Ji, Phong T Tran
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引用次数: 0

摘要

分裂酵母激酶-6 Klp9在后期定位于纺锤体中间区,产生滑动力以拉长双极纺锤体。在缺乏Klp9的情况下,后期纺锤体伸长率衰减为正常速率的一半。Klp9的功能是微管+端向四聚体马达。四聚化是其微管滑动功能的关键,因为四聚化允许Klp9在中间区域结合反平行的微管。Klp9的氨基酸序列显示两个α -螺旋螺旋结构域CC1和CC2,这两个结构域对蛋白质-蛋白质相互作用很重要。我们使用AlphaFold3通过Klp9的卷曲线圈寻找其潜在的寡聚化状态。AlphaFold预测CC1可以形成二聚体,并与CC2一起形成四聚体。不同的寡聚态使得精确的实验验证成为可能。我们测量了Klp9全长、Klp9缺失(Klp9Δ)和截断的Klp9不含卷曲线圈、只有CC1或CC1和CC2的Klp9马达GFP强度和后期纺锤伸长率。结果表明:1)GFP强度随着低聚体状态的增加而增加;2)仅在包含CC1和CC2的Klp9截断中恢复了衰减的后期纺锤体速度。实验数据与预测一致,表明CC1促进Klp9二聚化,并且CC1和CC2共同促进Klp9四聚化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
The two coiled-coil domains of the fission yeast kinesin-6 Klp9 are required for motor tetramerization and spindle elongation.

The fission yeast kinesin-6 Klp9 localizes to the spindle midzone at anaphase to produce sliding forces to elongate the bipolar spindle. In the absence of Klp9 , anaphase spindle elongation is attenuated by half its normal rate. Klp9 functions as a microtubule plus end-directed tetrameric motor. Tetramerization is the key to its microtubule sliding function, as tetramerization allows Klp9 to bind antiparallel microtubules at the midzone. The amino acid sequence of Klp9 indicates two alpha-helical coiled-coils domains CC1 and CC2, important for protein-protein interactions. We seeked the potential oligomerization states of Klp9 via its coiled-coils using AlphaFold3. AlphaFold predicted that CC1 can form dimers and together with CC2 can form tetramers. The different oligomeric states enabled precise experimental verifications. We measured Klp9 motor GFP intensity and anaphase spindle elongation rate for the full-length Klp9 , Klp9-deletion (Klp9Δ), and truncated Klp9 containing no coiled-coils, or only CC1, or both CC1 and CC2. The results indicate that: 1) GFP intensity increases with increasing oligomeric state, and 2) attenuated anaphase spindle velocity is restored only in the Klp9 truncation containing both CC1 and CC2. The experimental data are consistent with prediction, indicating that CC1 contributes to Klp9 dimerization, and that CC1 and CC2 together contribute to Klp9 tetramerization.

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