Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto 606-8502, Japan.
Exp Cell Res. 2012 Feb 1;318(3):262-75. doi: 10.1016/j.yexcr.2011.11.006. Epub 2011 Nov 15.
Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a +TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure.
Mal3 是裂殖酵母 EB1 的同源物，EB1 是一种微管末端追踪蛋白（+TIP）。我们生成了一个突变（89R），在 Mal3 的钙调蛋白同源（CH）结构域中将谷氨酰胺突变为精氨酸。体外分析 89R 突变体表明，该突变赋予微管更高的亲和力并增强了促进微管组装的固有活性。突变体 Mal3 不再是 +TIP，而是与微管晶格紧密结合。活细胞成像显示，尽管野生型 Mal3 蛋白在收缩开始前从生长中的微管末端解离，但突变体 Mal3 蛋白在微管上持续存在，并在更长的停顿后降低收缩速度。因此，突变体 Mal3 蛋白导致构成纺锤体和星体的微管异常伸长。Mal3 在连接 CH 和 EB1 样 C 端基序结构域的连接子上的一组丝氨酸/苏氨酸残基上被磷酸化。磷酸化以微管依赖性方式发生，并降低 Mal3 与微管的亲和力。我们提出，由于 89R 突变对磷酸化的作用具有抗性，因此它可以持续与微管结合，并通过增强圆柱状结构赋予微管更强的稳定性。
