Zhang Hui, Schmidt Bela Z, Sun Fei, Condliffe Steven B, Butterworth Michael B, Youker Robert T, Brodsky Jeffrey L, Aridor Meir, Frizzell Raymond A
Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 2006 Apr 21;281(16):11312-21. doi: 10.1074/jbc.M512013200. Epub 2006 Feb 9.
We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum.
我们研究了半胱氨酸串珠蛋白（Csp）在囊性纤维化跨膜传导调节因子（CFTR）生物合成中的作用，并将其与另一种J结构域蛋白Hdj-2（一种公认的CFTR辅助伴侣蛋白）进行了关联研究。Csp表达的增加导致成熟（C带）CFTR呈剂量依赖性减少，而未成熟（B带）CFTR增加。Hdj-2的外源性表达也增加了CFTR的B带，但与Csp不同的是，Hdj-2也增加了C带。Csp诱导的CFTR成熟阻滞需要Hsp70，因为一个干扰Csp刺激Hsp70 ATP酶活性能力的J结构域突变体（H43Q）缓解了Csp诱导的CFTR成熟阻滞。然而，Csp H43Q仍然增加了未成熟的CFTR。发现Csp诱导的B带CFTR位于细胞核附近，与钙连接蛋白共定位，并且它仍然可溶于去污剂。这些数据表明，Csp不是通过促进未成熟CFTR的聚集或降解来阻滞CFTR成熟。通过RNA干扰敲低Csp导致成熟CFTR增加了5倍，并增强了cAMP刺激的CFTR电流。因此，成熟CFTR的产生与Csp的表达水平呈负相关。Csp和Hdj-2在体外均与CFTR的R结构域相关联，并且Csp取代了Hdj-2的结合，这表明它们具有共同的相互作用位点。Csp和Hdj-2的联合表达模拟了单独Csp的作用，即阻滞CFTR成熟。但是，Csp和Hdj-2一起使CFTR的B带呈累加性增加，并且这并不取决于它们与Hsp70的相互作用，这与这些蛋白的直接伴侣作用一致。与Hdj-2一样，在没有Hsp70的情况下，Csp在体外减少了NBD1的聚集。我们的数据表明，Csp和Hdj-2都促进未成熟CFTR的生物合成，作为直接的CFTR伴侣蛋白，但此外，Csp在CFTR生物合成级联中位于较后的位置，在那里它通过限制成熟CFTR从内质网的输出量来调节其产生。
