Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
J Cell Sci. 2011 Sep 15;124(Pt 18):3164-73. doi: 10.1242/jcs.087320.
The UNC-45 family of molecular chaperones is expressed in metazoan organisms from Caenorhabditis elegans to humans. The UNC-45 protein is essential in C. elegans for early body-wall muscle cell development and A-band assembly. We show that the myosin-binding UCS domain of UNC-45 alone is sufficient to rescue lethal unc-45 null mutants arrested in embryonic muscle development and temperature-sensitive loss-of-function unc-45 mutants defective in worm A-band assembly. Removal of the Hsp90-binding TPR domain of UNC-45 does not affect rescue. Similar results were obtained with overexpression of the same fragments in wild-type nematodes when assayed for diminution of myosin accumulation and assembly. Titration experiments show that, on a per molecule basis, UCS has greater activity in C. elegans muscle in vivo than full-length UNC-45 protein, suggesting that UNC-45 is inhibited by either the TPR domain or its interaction with the general chaperone Hsp90. In vitro experiments with purified recombinant C. elegans Hsp90 and UNC-45 proteins show that they compete for binding to C. elegans myosin. Our in vivo genetic and in vitro biochemical experiments are consistent with a novel inhibitory role for Hsp90 with respect to UNC-45 action.
UNC-45 分子伴侣家族在从秀丽隐杆线虫到人类的后生动物中表达。UNC-45 蛋白在秀丽隐杆线虫中对于早期体壁肌肉细胞发育和 A 带组装是必不可少的。我们表明，UNC-45 的肌球蛋白结合 UCS 结构域本身就足以挽救在胚胎肌肉发育中被阻断的致命 unc-45 缺失突变体和在 A 带组装中功能丧失的温度敏感 unc-45 突变体。UNC-45 的 Hsp90 结合 TPR 结构域的缺失并不影响拯救。在野生型线虫中过表达相同片段时，也获得了类似的结果，用于检测肌球蛋白积累和组装的减少。滴定实验表明，基于每个分子，UCS 在秀丽隐杆线虫肌肉中的活性高于全长 UNC-45 蛋白，这表明 UNC-45 受到 TPR 结构域或其与一般伴侣 Hsp90 的相互作用的抑制。用纯化的重组秀丽隐杆线虫 Hsp90 和 UNC-45 蛋白进行的体外实验表明，它们竞争结合秀丽隐杆线虫肌球蛋白。我们的体内遗传和体外生化实验与 Hsp90 对 UNC-45 作用的新的抑制作用一致。
