Department of Molecular Cellular Biology, University of California, Davis, Davis, CA 95616.
Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143.
Mol Biol Cell. 2019 Jun 1;30(12):1490-1504. doi: 10.1091/mbc.E19-02-0093. Epub 2019 Apr 10.
XMAP215/Stu2/Alp14 accelerates tubulin polymerization while processively tracking microtubule (MT) plus ends via tumor overexpressed gene (TOG) domain arrays. It remains poorly understood how these functions arise from tubulin recruitment, mediated by the distinct TOG1 and TOG2 domains, or the assembly of these arrays into large square complexes. Here, we describe a relationship between MT plus-end tracking and polymerase functions revealing their distinct origin within TOG arrays. We study Alp14 mutants designed based on structural models, with defects in either tubulin recruitment or self-organization. Using in vivo live imaging in fission yeast and in vitro MT dynamics assays, we show that tubulins recruited by TOG1 and TOG2 serve concerted, yet distinct, roles in MT plus-end tracking and polymerase functions. TOG1 is critical for processive plus-end tracking, whereas TOG2 is critical for accelerating tubulin polymerization. Inactivating interfaces that stabilize square complexes lead to defects in both processive MT plus-end tracking and polymerase. Our studies suggest that a dynamic cycle between square and unfurled TOG array states gives rise to processive polymerase activity at MT plus ends.
XMAP215/Stu2/Alp14 通过肿瘤过表达基因 (TOG) 结构域阵列加速微管 (MT) 聚合，同时进行性追踪 MT 末端。目前还不太清楚这些功能是如何通过由独特的 TOG1 和 TOG2 结构域介导的微管募集，或这些阵列组装成大的正方形复合物产生的。在这里，我们描述了 MT 末端追踪和聚合酶功能之间的关系，揭示了它们在 TOG 阵列中的不同起源。我们研究了基于结构模型设计的 Alp14 突变体，这些突变体在微管募集或自组装方面存在缺陷。我们使用裂殖酵母体内实时成像和体外 MT 动力学测定，表明由 TOG1 和 TOG2 募集的微管在 MT 末端追踪和聚合酶功能方面协同发挥作用，但具有不同的作用。TOG1 对于进行性末端追踪至关重要，而 TOG2 对于加速微管聚合至关重要。失活稳定正方形复合物的界面会导致进行性 MT 末端追踪和聚合酶功能缺陷。我们的研究表明，正方形和展开的 TOG 阵列状态之间的动态循环导致 MT 末端进行性聚合酶活性。
