Activation of the γ-Tubulin Complex by the Mto1/2 Complex
Activation of the γ-Tubulin Complex by the Mto1/2 Complex
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DOI：
10.1016/j.cub.2014.03.006
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发表时间：
2014-04-14
期刊：
影响因子：
9.2
通讯作者：
Sawin, Kenneth E.
中科院分区：
文献类型：
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作者：
Lynch, Eric M.;Groocock, Lynda M.;Sawin, Kenneth E.
多亚基γ-微管蛋白复合物（γ-TuC）对于真核细胞中的微管成核至关重要[1，2]，但仍不清楚γ-TuC如何在微管组织中心（MTOC）特异性地变得活跃，而不是在整个细胞质中更广泛地活跃[3，4]。在裂殖酵母粟酒裂殖酵母中，蛋白质Mto 1和Mto 2形成Mto 1/2复合物，该复合物与γ-TuC相互作用并将其招募到几种不同类型的细胞质MTOC位点[5-10]。在这里，我们表明，Mto 1/2复合物激活γ-TuC依赖的微管成核独立的本地化的γ-TuC。这是通过构建Mto 1/2的“最小”版本Mto 1/2[bonsai]来实现的，该版本不本地化到任何MTOC站点。通过直接成像的个别Mto 1/2[盆景]复合物成核单个微管在体内，我们进一步确定的数量和化学计量的Mto 1，Mto 2，和γ-TuC亚基Alp 4（GCP 2）和Alp 6（GCP 3）内的活性成核复合物。这些结果与每个活性复合物含有类似于13个拷贝的Mto 1和Mto 2的活性成核复合物和可能等摩尔量的γ-微管蛋白一致。另外的实验表明，Mto 1/2多聚体的作用是使裂变酵母γ-微管蛋白小复合物多聚化，特别是Mto 2的多聚化可能是活性微管成核复合物组装的基础。
The multisubunit gamma-tubulin complex (gamma-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the gamma-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the gamma-TuC and recruits it to several different types of cytoplasmic MTOC sites [5-10]. Here, we show that the Mto1/2 complex activates gamma-TuC-dependent microtubule nucleation independently of localizing the gamma-TuC. This was achieved through the construction of a "minimal'' version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and gamma-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing similar to 13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of gamma-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast gamma-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes.