Tian G, Lewis S A, Feierbach B, Stearns T, Rommelaere H, Ampe C, Cowan N J
Department of Biochemistry, New York University Medical Center 10016, USA.
J Cell Biol. 1997 Aug 25;138(4):821-32. doi: 10.1083/jcb.138.4.821.
The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.
天然α/β微管蛋白异二聚体的体外产生依赖于胞质伴侣蛋白和几种蛋白质辅因子的作用。我们之前表明，四种这样的辅因子（称为A、C、D和E）与天然微管蛋白一起作用于伴侣蛋白产生的β微管蛋白折叠中间体，以产生可聚合的微管蛋白异二聚体。然而，这组辅因子从同一伴侣蛋白产生的α微管蛋白折叠中间体中产生天然异二聚体的效率非常低。在这里，我们描述了一种新型微管蛋白折叠辅因子（辅因子B）的分离、表征和遗传分析，它在体外极大地提高了α微管蛋白折叠的效率。这使得对α和β微管蛋白折叠进行综合研究成为可能：我们发现，导致天然α和β微管蛋白形成的途径是趋同的，因为α亚基的折叠需要含有β亚基的辅因子复合物的参与，反之亦然。我们还表明，通过与辅因子形成复合物来隔离天然α或β微管蛋白会导致其余游离亚基的不稳定和降解。这些数据表明，微管蛋白折叠辅因子通过将α和β微管蛋白多肽置于和/或维持在形成天然α/β异二聚体所需的活化构象状态来发挥作用，这意味着每个亚基为另一个亚基的正确折叠提供了必要的信息。
