Luchniak Anna, Fukuda Yusuke, Gupta Mohan L
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
Methods Cell Biol. 2013;115:355-74. doi: 10.1016/B978-0-12-407757-7.00022-0.
Microtubules play essential roles in a wide variety of cellular processes including cell division, motility, and vesicular transport. Microtubule function depends on the polymerization dynamics of tubulin and specific interactions between tubulin and diverse microtubule-associated proteins. To date, investigation of the structural and functional properties of tubulin and tubulin mutants has been limited by the inability to obtain functional protein from overexpression systems, and by the heterogeneous mixture of tubulin isotypes typically isolated from higher eukaryotes. The budding yeast, Saccharomyces cerevisiae, has emerged as a leading system for tubulin structure-function analysis. Yeast cells encode a single beta-tubulin gene and can be engineered to express just one of two alpha isotypes. Moreover, yeast allows site-directed modification of tubulin genes at the endogenous loci expressed under the native promoter and regulatory elements. These advantageous features provide a homogeneous and controlled environment for analysis of the functional consequences of specific mutations. Here, we present the techniques to generate site-specific tubulin mutations in diploid and haploid cells, assess the ability of the mutated protein to support cell viability, measure overall microtubule stability, and define changes in the specific parameters of microtubule dynamic instability. We also outline strategies to determine whether mutations disrupt interactions with microtubule-associated proteins. Microtubule-based functions in yeast are well defined, which allows the observed changes in microtubule properties to be related to the role of microtubules in specific cellular processes.
微管在包括细胞分裂、运动及囊泡运输等多种细胞过程中发挥着重要作用。微管功能取决于微管蛋白的聚合动力学以及微管蛋白与多种微管相关蛋白之间的特定相互作用。迄今为止，对微管蛋白及其突变体的结构和功能特性的研究受到限制，一方面无法从过表达系统中获得功能性蛋白，另一方面通常从高等真核生物中分离得到的微管蛋白亚型存在异质性混合物。出芽酵母酿酒酵母已成为微管蛋白结构 - 功能分析的主要系统。酵母细胞编码单个β - 微管蛋白基因，并且可以进行工程改造以仅表达两种α亚型之一。此外，酵母允许在天然启动子和调控元件控制下对内源基因座处的微管蛋白基因进行定点修饰。这些有利特性为分析特定突变的功能后果提供了一个均匀且可控的环境。在此，我们介绍在二倍体和单倍体细胞中产生位点特异性微管蛋白突变的技术，评估突变蛋白支持细胞活力的能力，测量整体微管稳定性，并确定微管动态不稳定性特定参数的变化。我们还概述了确定突变是否破坏与微管相关蛋白相互作用的策略。酵母中基于微管的功能已得到很好的定义，这使得观察到的微管特性变化能够与微管在特定细胞过程中的作用相关联。
