Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum-associated degradation.
Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum-associated degradation.
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DOI：
10.1091/mbc.e11-02-0137
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发表时间：
2011-08-15
影响因子：
3.3
通讯作者：
Skach WR
中科院分区：
文献类型：
--
作者：
Matsumura Y;David LL;Skach WR
Hsc70在共译性囊性纤维化跨膜传导调节因子折叠过程中发挥了生产作用，其在翻译后靶向泛素-蛋白酶体系统中的主要贡献超过了这一作用。此外，Hsc70结合的结果似乎对其结合周期的持续时间高度敏感，这是由调节伴侣控制的。Hsp/c70细胞质伴侣系统促进了蛋白质折叠和降解的竞争途径。在这里，我们使用重建的无细胞系统来研究Hsc70在膜蛋白囊性纤维化跨膜传导调节因子（CFTR）的共同和翻译后决策中的作用机制和程度。Hsc70与CFTR的结合被Bag-1的c端结构域（CBag）破坏，通过加速ADP-ATP交换刺激client释放。在CFTR翻译过程中加入CBag略微增加了新合成蛋白的降解易感性，这与Hsc70的轮廓功能一致。相反，Hsc70结合的翻译后不稳定几乎完全阻断了CFTR的泛素化、内质网脱位和蛋白酶体介导的裂解。这种效应需要CBag相对于Hsc70的摩尔过量，并且被Hsc70的CBag结合子结构域完全逆转。这些结果表明，Hsc70在共翻译CFTR折叠过程中的预折叠作用被翻译后靶向泛素-蛋白酶体系统的主导和重要作用所抵消。此外，Hsc70结合的降解结果似乎对其结合周期的持续时间高度敏感，而这反过来又受到调节伴侣的整合表达的控制。
Hsc70 plays a productive role during cotranslational cystic fibrosis transmembrane conductance regulator folding that is outweighed by its dominant contribution to posttranslational targeting to the ubiquitin-proteasome system. Moreover, the outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is governed by regulatory cochaperones. The Hsp/c70 cytosolic chaperone system facilitates competing pathways of protein folding and degradation. Here we use a reconstituted cell-free system to investigate the mechanism and extent to which Hsc70 contributes to these co- and posttranslational decisions for the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR). Hsc70 binding to CFTR was destabilized by the C-terminal domain of Bag-1 (CBag), which stimulates client release by accelerating ADP-ATP exchange. Addition of CBag during CFTR translation slightly increased susceptibility of the newly synthesized protein to degradation, consistent with a profolding function for Hsc70. In contrast, posttranslational destabilization of Hsc70 binding nearly completely blocked CFTR ubiquitination, dislocation from the endoplasmic reticulum, and proteasome-mediated cleavage. This effect required molar excess of CBag relative to Hsc70 and was completely reversed by the CBag-binding subdomain of Hsc70. These results demonstrate that the profolding role of Hsc70 during cotranslational CFTR folding is counterbalanced by a dominant and essential role in posttranslational targeting to the ubiquitin-proteasome system. Moreover, the degradative outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is in turn governed by the integrated expression of regulatory cochaperones.