Tian Guoling, Cowan Nicholas J
Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, New York, USA.
Methods Cell Biol. 2013;115:155-71. doi: 10.1016/B978-0-12-407757-7.00011-6.
The tubulin heterodimer consists of one α- and one β-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones (TBCs) termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native β- and α-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of β-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/β and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCE/α/TBCD/β). Discharge of the native α/β heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound β-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of E-site GDP-bound heterodimer, which becomes polymerization competent following spontaneous exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and β-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or β-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a nonminimal energy state. Here, we describe methods for the purification of recombinant TBCs as biologically active proteins following their expression in a variety of host/vector systems.
微管蛋白异二聚体由一个α-微管蛋白和一个β-微管蛋白多肽组成。如果没有包括五个微管蛋白特异性伴侣蛋白（TBCs，称为TBCA - TBCE）在内的一系列伴侣蛋白的协同作用，这两种蛋白质都无法形成天然状态，也无法组装成具有聚合能力的异二聚体。在通过与胞质伴侣蛋白进行多轮ATP依赖的相互作用产生新合成的准天然β-和α-微管蛋白多肽后，TBCA和TBCB分别与之结合并使其稳定。β-微管蛋白在TBCA和TBCD之间自由交换，α-微管蛋白在TBCB和TBCE之间自由交换，分别形成TBCD/β和TBCE/α。后两种复合物相互作用，形成一个超复合物（TBCE/α/TBCD/β）。天然α/β异二聚体的释放是通过超复合物与TBCC的相互作用实现的，这导致与TBC结合的β-微管蛋白（E位点）的GTP水解。该反应作为超复合物解离和E位点结合GDP的异二聚体释放（与GTP自发交换后变得具有聚合能力）的开关。因此，微管蛋白特异性伴侣蛋白共同作为微管蛋白组装机器发挥作用，将α-和β-微管蛋白亚基结合成紧密相连的异二聚体。这种进化上保守的途径的存在解释了为什么在没有其同源伴侣的情况下，从未成功地将α-或β-微管蛋白分离为稳定的独立实体，这意味着它们各自以非最小能量状态存在并维持在异二聚体中。在这里，我们描述了在各种宿主/载体系统中表达后，将重组TBCs纯化为具有生物活性蛋白质的方法。
