The Thiazide-sensitive NaCl Cotransporter Is Targeted for Chaperone-dependent Endoplasmic Reticulum-associated Degradation
The Thiazide-sensitive NaCl Cotransporter Is Targeted for Chaperone-dependent Endoplasmic Reticulum-associated Degradation
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DOI：
10.1074/jbc.m111.288928
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发表时间：
2011-12-23
影响因子：
4.8
通讯作者：
Brodsky, Jeffrey L.
中科院分区：
文献类型：
--
作者：
Needham, Patrick G.;Mikoluk, Kasia;Brodsky, Jeffrey L.
噻嗪敏感性NaCl协同转运蛋白（NCC，SLC12A3）介导肾脏远端肾单位中的盐重吸收，并且是噻嗪类利尿剂的靶点，噻嗪类利尿剂通常用于治疗高血压。NCC中的突变也引起Gitelman综合征，这是一种遗传性盐耗障碍，在大多数情况下被认为是通过增强的内质网相关降解（ERAD）由受损的NCC生物发生引起的。因为介导NCC质量控制的机制是完全不确定的，我们采用酵母作为模型异源表达系统，以确定参与NCC降解的因素。我们证实NCC是酵母中真正的ERAD底物，因为NCC多肽的大部分整合到ER膜中，并且其周转率对蛋白酶体抑制敏感。NCC降解主要依赖于ER膜相关的E3泛素连接酶Hrd1。而几个ER管腔伴侣NCC ERAD，NCC泛素化和降解需要的活性Ssa 1，细胞质Hsp70伴侣。当NCC在哺乳动物肾细胞中表达时，观察到相容的发现，因为协同转运蛋白被蛋白酶体多聚泛素化和降解，并且哺乳动物细胞质Hsp70（Hsp72）共表达刺激新合成的NCC的降解。Hsp70也优先与ER定位的NCC糖基化种类相关，表明细胞质Hsp70在选择ERAD的NCC的不成熟形式中起关键作用。总之，这些结果提供了第一次调查的组成部分参与ERAD的哺乳动物SLC12阳离子氯共转运蛋白，并提供了一个框架，为今后的研究NCC ER质量控制。
The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation. We confirmed that NCC was a bona fide ERAD substrate in yeast, as the majority of NCC polypeptide was integrated into ER membranes, and its turnover rate was sensitive to proteasome inhibition. NCC degradation was primarily dependent on the ER membrane-associated E3 ubiquitin ligase Hrd1. Whereas several ER luminal chaperones were dispensable for NCC ERAD, NCC ubiquitination and degradation required the activity of Ssa1, a cytoplasmic Hsp70 chaperone. Compatible findings were observed when NCC was expressed in mammalian kidney cells, as the cotransporter was polyubiquitinated and degraded by the proteasome, and mammalian cytoplasmic Hsp70 (Hsp72) coexpression stimulated the degradation of newly synthesized NCC. Hsp70 also preferentially associated with the ER-localized NCC glycosylated species, indicating that cytoplasmic Hsp70 plays a critical role in selecting immature forms of NCC for ERAD. Together, these results provide the first survey of components involved in the ERAD of a mammalian SLC12 cation chloride cotransporter and provide a framework for future studies on NCC ER quality control.