Dionne M A, Sanchez A, Compton D A
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 2000 Apr 21;275(16):12346-52. doi: 10.1074/jbc.275.16.12346.
Microtubules induced to polymerize with taxol in a mammalian mitotic extract organize into aster-like arrays in a centrosome-independent process that is driven by microtubule motors and structural proteins. These microtubule asters accurately reflect the noncentrosomal aspects of mitotic spindle pole formation. We show here that colonic-hepatic tumor-overexpressed gene (ch-TOGp) is an abundant component of these asters. We have prepared ch-TOGp-specific antibodies and show by immunodepletion that ch-TOGp is required for microtubule aster assembly. Microtubule polymerization is severely inhibited in the absence of ch-TOGp, and silver stain analysis of the ch-TOGp immunoprecipitate indicates that it is not present in a preformed complex and is the only protein removed from the extract during immunodepletion. Furthermore, the reduction in microtubule polymerization efficiency in the absence of ch-TOGp is dependent on ATP. These results demonstrate that ch-TOGp is a major constituent of microtubule asters assembled in a mammalian mitotic extract and that it is required for robust microtubule polymerization in an ATP-dependent manner in this system even though taxol is present. These data, coupled with biochemical and genetic data derived from analysis of ch-TOGp-related proteins in other organisms, indicate that ch-TOGp is a key factor regulating microtubule dynamics during mitosis.
在哺乳动物有丝分裂提取物中，用紫杉醇诱导聚合的微管在一个由微管马达和结构蛋白驱动的、不依赖中心体的过程中组织成星状阵列。这些微管星状体准确反映了有丝分裂纺锤体极形成的非中心体方面。我们在此表明，结肠-肝肿瘤过表达基因（ch-TOGp）是这些星状体的一个丰富成分。我们制备了ch-TOGp特异性抗体，并通过免疫去除实验表明，微管星状体组装需要ch-TOGp。在没有ch-TOGp的情况下，微管聚合受到严重抑制，对ch-TOGp免疫沉淀物的银染分析表明，它不存在于预先形成的复合物中，并且是免疫去除过程中从提取物中唯一去除的蛋白质。此外，在没有ch-TOGp的情况下，微管聚合效率的降低依赖于ATP。这些结果表明，ch-TOGp是在哺乳动物有丝分裂提取物中组装的微管星状体的主要成分，并且即使存在紫杉醇，在该系统中它也是以ATP依赖的方式进行强大微管聚合所必需的。这些数据，再加上从分析其他生物体中与ch-TOGp相关的蛋白质获得的生化和遗传数据，表明ch-TOGp是有丝分裂期间调节微管动力学的关键因素。
