The bile salt export pump (BSEP) is a pivotal apical/canalicular bile salt transporter in hepatocytes that drives the bile flow. Defects in BSEP function and canalicular expression could lead to a spectrum of cholestatic liver diseases. One prominent manifestation of BSEP-associated cholestasis is the defective canalicular localization and cytoplasmic retention of BSEP. However, the etiology of impaired BSEP targeting to the canalicular membrane is not fully understood. Our goal was to discover what molecule could interact with BSEP and affect its post-Golgi sorting. The human BSEP amino acids (a.a.) 491-630 was used as bait to screen a human fetal liver cDNA library through yeast two-hybrid system. We identified a BSEP-interacting candidate and showed the interaction and colocalization in the co-immunoprecipitation in hepatoma cell lines and histological staining in human liver samples. Temperature shift assays were used to study the post-Golgi trafficking of BSEP. We further determine the functional impacts of the BSEP-interacting candidate on BSEP in vitro. A hydrodynamically injected mouse model was established for in vivo characterizing the long-term impacts on BSEP. We identified that charged multivesicular body protein 5 (CHMP5), a molecule of the endosomal protein complex required for transport subcomplex-III (ESCRT-III), interacted and co-localized with BSEP in the subapical compartments (SACs) in developing human livers. Cholestatic BSEP mutations in the CHMP5-interaction region have defects in canalicular targeting and aberrant retention at the SACs. Post-Golgi delivery of BSEP and bile acid secretion were impaired in ESCRT-III perturbation or CHMP5-knockdown hepatic cellular and mouse models. This ESCRT-III-mediated BSEP sorting preceded Rab11A-regulated apical cycling of BSEP. Our results showed the first example that ESCRT-III is essential for canalicular trafficking of apical membrane proteins, and provide new targets for therapeutic approaches in BSEP associated cholestasis.

中文翻译：

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ESCRT-III 分子调节胆盐输出泵的顶端靶向

胆盐输出泵 (BSEP) 是肝细胞中驱动胆汁流动的关键顶端/小管胆汁盐转运蛋白。BSEP 功能和小管表达的缺陷可能导致一系列胆汁淤积性肝病。BSEP 相关胆汁淤积的一个突出表现是 BSEP 的小管定位和细胞质滞留缺陷。然而，靶向小管膜的 BSEP 受损的病因尚不完全清楚。我们的目标是发现什么分子可以与 BSEP 相互作用并影响其后高尔基体分选。以人BSEP氨基酸（aa）491-630为诱饵，通过酵母双杂交系统筛选人胎肝cDNA文库。我们确定了一个 BSEP 相互作用候选物，并显示了肝癌细胞系中免疫共沉淀和人肝脏样本组织学染色中的相互作用和共定位。温度变化测定用于研究高尔基体后 BSEP 的运输。我们进一步确定了 BSEP 相互作用候选物对 BSEP 体外 的功能影响。建立了流体动力学注射的小鼠模型，用于在体内表征对 BSEP 的长期影响。我们发现带电多泡体蛋白 5 (CHMP5)，一种转运亚复合物 III (ESCRT-III) 所需的内体蛋白复合物分子，在人类肝脏发育过程中与根尖下隔室 (SAC) 中的 BSEP 相互作用并共定位。CHMP5 相互作用区域中的胆汁淤积 BSEP 突变在小管靶向和 SAC 的异常保留方面存在缺陷。高尔基体后 BSEP 和胆汁酸分泌在 ESCRT-III 扰动或 CHMP5 敲低肝细胞和小鼠模型中受损。这种 ESCRT-III 介导的 BSEP 分选先于 Rab11A 调节的 BSEP 顶端循环。我们的结果显示了第一个例子，即 ESCRT-III 对顶端膜蛋白的小管运输至关重要，并为 BSEP 相关胆汁淤积的治疗方法提供了新的靶点。