Institute of Biochemistry, Eidgenössische Technische Hochschule Zürich , Zurich, Switzerland.
CRBM, Université de Montpellier , CNRS, Montpellier, France.
J Cell Biol. 2023 Jul 3;222(7). doi: 10.1083/jcb.202110126. Epub 2023 Apr 24.
Kinesins are microtubule-dependent motor proteins, some of which moonlight as microtubule polymerases, such as the yeast protein Kip2. Here, we show that the CLIP-170 ortholog Bik1 stabilizes Kip2 at microtubule ends where the motor domain of Kip2 promotes microtubule polymerization. Live-cell imaging and mathematical estimation of Kip2 dynamics reveal that disrupting the Kip2-Bik1 interaction aborts Kip2 dwelling at microtubule ends and abrogates its microtubule polymerization activity. Structural modeling and biochemical experiments identify a patch of positively charged residues that enables the motor domain to bind free tubulin dimers alternatively to the microtubule shaft. Neutralizing this patch abolished the ability of Kip2 to promote microtubule growth both in vivo and in vitro without affecting its ability to walk along microtubules. Our studies suggest that Kip2 utilizes Bik1 as a cofactor to track microtubule tips, where its motor domain then recruits free tubulin and catalyzes microtubule assembly.
驱动蛋白是微管依赖性的马达蛋白，其中一些蛋白兼有微管聚合酶的功能，例如酵母蛋白 Kip2。在这里，我们发现 CLIP-170 同源物 Bik1 稳定 Kip2 在微管末端，而 Kip2 的马达结构域促进微管聚合。活细胞成像和对 Kip2 动力学的数学估计表明，破坏 Kip2-Bik1 相互作用会终止 Kip2 在微管末端的停留，并取消其微管聚合活性。结构建模和生化实验确定了一个带正电荷的残基，使马达结构域能够替代地结合游离的微管二聚体到微管轴上。中和该区域会破坏 Kip2 在体内和体外促进微管生长的能力，而不影响其沿微管行走的能力。我们的研究表明，Kip2 利用 Bik1 作为辅助因子来追踪微管末端，然后其马达结构域招募游离的微管蛋白并催化微管组装。
