Microtubule nucleation is spatiotemporally regulated in cells by several molecules, including the template γ-tubulin and the polymerase XMAP215. Recently, XMAP215 and the γ-tubulin ring complex were reported to function synergistically, and this synergy was hypothesized to be due to direct binding between XMAP215 and γ-tubulin. Here, we address this hypothesis by 1) probing domain requirements for XMAP215 to promote microtubule nucleation and 2) testing whether XMAP215 functions synergistically with γ-tubulin in the absence of the other ring complex proteins. We confirm that γ-tubulin and XMAP215 are classically defined nucleators that reduce the nucleation lag seen in bulk tubulin assembly. Then, using deletion constructs, we show that XMAP215's ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and γ-tubulin promote αβ-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct, and the synergy reported between XMAP215 and the γ-tubulin ring complex is not due to γ-tubulin alone.

中文翻译：

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XMAP215和γ-微管蛋白可在纯化溶液中加成促进微管成核

细胞中的微管成核在时空上受几个分子的调节，包括模板γ-微管蛋白和聚合酶XMAP215。最近，据报道XMAP215和γ-微管蛋白环复合物具有协同作用，并且推测这种协同作用是由于XMAP215和γ-微管蛋白之间的直接结合。在这里，我们通过以下方法解决这一假设：1）探索XMAP215的结构域要求以促进微管成核，以及2）在不存在其他环复合蛋白的情况下测试XMAP215是否与γ-微管蛋白协同作用。我们确认，γ-微管蛋白和XMAP215是经典定义的成核剂，可减少在批量微管蛋白装配中看到的成核滞后。然后，使用删除结构显示XMAP215' 在纯化溶液中使微管成核的能力与其拉长现有微管的能力有关，并且不取决于TOG域的数量。最后，我们显示XMAP215和γ-微管蛋白以加性而非协同方式促进α-微管蛋白装配。因此，它们在微管成核过程中的作用方式是不同的，并且XMAP215和γ-微管蛋白环复合物之间的协同作用并非仅由于γ-微管蛋白引起。