Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco
Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco
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DOI：
10.1104/pp.15.01173
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发表时间：
2016-03-01
期刊：
影响因子：
7.4
通讯作者：
Hashimoto, Takashi
中科院分区：
文献类型：
--
作者：
Hotta, Takashi;Fujita, Satoshi;Hashimoto, Takashi
微管组装成几个不同的阵列，在细胞分裂和细胞形态发生中发挥重要作用。要破译的机制，调节这种多功能的细胞骨架成分的动态和组织，这是必不可少的，以建立在体外试验，使用功能微管蛋白。虽然植物微管蛋白已被纯化从原生质体的可逆紫杉醇诱导聚合，一个简单而有效的纯化方法尚未开发。在这里，我们使用了肿瘤过表达基因（TOG）列，其中的微管蛋白结合结构域的酵母（酿酒酵母）TOG同系物固定在树脂上，分离功能植物微管蛋白。我们发现，几百微克的纯微管蛋白可以很容易地从烟草（烟草）和拟南芥（拟南芥）的细胞悬浮培养物中纯化。通过TOG柱纯化的微管蛋白显示出高的组装能力，部分原因是α-微管蛋白的聚合抑制磷酸化水平低。与猪脑微管蛋白相比，拟南芥微管蛋白在体外的正负两端都是高度动态的，表现出更快的收缩速率和更频繁的灾难事件，并表现出频繁的自发成核。此外，我们的研究表明，α-微管蛋白中的内部组氨酸标签可用于制备特定同种型和特异性工程化的α-微管蛋白。与以前的研究植物微管蛋白，我们的质谱和免疫印迹分析未能检测到分离的拟南芥微管蛋白的翻译后修饰或检测到只有低水平的翻译后修饰。这种新技术可用于从植物细胞培养物中制备具有组装能力的、高度动态的纯微管蛋白。
Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of alpha-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in a-tubulin can be used to prepare particular isotypes and specifically engineered versions of alpha-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.