An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue.
An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue.
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一个孤立的扣子TOG域抑制微管灾难并促进救援。
DOI：
10.1091/mbc.e17-12-0748
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发表时间：
2018-06-01
影响因子：
3.3
通讯作者：
Rice LM
中科院分区：
文献类型：
--
作者：
Majumdar S;Kim T;Chen Z;Munyoki S;Tso SC;Brautigam CA;Rice LM
微管是受到严格调控的αβ-微管蛋白的动态聚合物，是染色体正确分离和细胞质组织所必需的。XMAP215家族中的聚合酶使用排列的TOG结构域来促进更快的微管延伸。细胞质连接物相关蛋白（CLASP）家族中减少灾难和/或增加救援的调节因子也含有排列的TOGs，但对CLASP TOGs如何促进活性知之甚少。本文以酿酒酵母Stu1为模型CLASP，进行了结构、生化和重构研究，阐明了CLASP TOGs的功能特性。Stu1中的两个TOGs具有非常不同的微管蛋白结合特性：TOG2与未聚合和聚合的微管蛋白结合，而TOG1与两者的结合都非常弱。Stu1-TOG2的结构揭示了一个clasp特异性残基，可能赋予了独特的微管蛋白结合特性。分离的TOG2结构域在体外强烈抑制微管突变并增加微管拯救，这与认为调控活动需要一系列tog的预期相矛盾。TOG2微管蛋白结合表面的单点突变会破坏其体外抗灾难和救援活性，并破坏Stu1在细胞中的功能。揭示一个分离的CLASP TOG可以调节聚合动力学而不作为阵列的一部分，这为CLASP的机制提供了新的见解，并丰富了对TOG功能的理解。
Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.