Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle.
Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle.
复制标题
DOI：
10.1242/bio.022905
复制
发表时间：
2017-05-15
期刊：
影响因子：
2.4
通讯作者：
Wakefield JG
中科院分区：
文献类型：
--
作者：
Chen JWC;Chen ZA;Rogala KB;Metz J;Deane CM;Rappsilber J;Wakefield JG
异八聚体蛋白复合体AUGMIN将γ-微管蛋白环复合体(γ-TURC)招募到预先存在的微管(MT)上，在有丝分裂期间产生分枝的微管(MT)，促进纺锤体的强健组装。然而，尽管最近在体外部分重建了人类Augmin复合体，但这种招募的分子基础仍然不清楚。在这里，我们使用免疫亲和纯化的体内果蝇Augmin和交联/质谱仪来确定在没有任何其他结构信息的情况下，8个Augmin亚基内残基之间的距离限制。结果使我们能够预测AUGMIN和γ-TURC之间的潜在界面。我们在果蝇胚胎中验证了这些预测，表明Augmin亚单位的特定区域Dgt3、Dgt5和Dgt6都直接与γ-TURC蛋白Dgp71WD结合，并且是γ-TURC积累所必需的，但不是AUGMIN与有丝分裂纺锤体的结合。因此，这项研究大大增加了我们对MT依赖的MT成核的分子机制的理解。摘要：果蝇的亚基位置限制图显示Dgt3、Dgt5和Dgt6都是有丝分裂纺锤体定位所必需的。
The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex in vitro, the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification of in vivo Augmin from Drosophila and cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information. The results allowed us to predict potential interfaces between Augmin and γ-TuRC. We tested these predictions biochemically and in the Drosophila embryo, demonstrating that specific regions of the Augmin subunits, Dgt3, Dgt5 and Dgt6 all directly bind the γ-TuRC protein, Dgp71WD, and are required for the accumulation of γ-TuRC, but not Augmin, to the mitotic spindle. This study therefore substantially increases our understanding of the molecular mechanisms underpinning MT-dependent MT nucleation. Summary: A map of subunit positional restrictions in Drosophila Augmin shows that Dgt3, Dgt5 and Dgt6 are all required to localise gamma-TuRC to mitotic spindles.