三聚体 Rab7 GEF 控制 NPC1 依赖性溶酶体胆固醇输出。
A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.
机构信息
Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK.
Leiden University Medical Centre, Leiden University, Leiden, The Netherlands.
出版信息
Nat Commun. 2020 Nov 3;11(1):5559. doi: 10.1038/s41467-020-19032-0.
Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.
胆固醇在哺乳动物细胞中的摄取是通过 LDL 受体途径介导的。在这里，我们使用内源性胆固醇报告基因进行了全基因组 CRISPR 筛选，鉴定出了 100 多个参与 LDL 胆固醇摄取的基因。我们将 C18orf8 鉴定为哺乳动物 Mon1-Ccz1 鸟嘌呤核苷酸交换因子（GEF）的核心亚基，该因子对于 Rab7 的复合物稳定性和功能至关重要。C18orf8 缺陷细胞缺乏 Rab7 的激活，并且在内体晚期(endosome)形态和内体 LDL 运输方面表现出严重缺陷，导致细胞胆固醇缺乏。出乎意料的是，游离胆固醇在肿胀的溶酶体中积累，表明溶酶体胆固醇输出存在严重缺陷。我们发现，活性 Rab7 与 NPC1 胆固醇转运蛋白相互作用，并许可溶酶体胆固醇输出。在 C18orf8、Ccz1 和 Mon1A/B 缺陷细胞中，该过程被废除，而通过组成型激活的 Rab7 则可以恢复该过程。因此，三聚体 Mon1-Ccz1-C18orf8 (MCC) GEF 在协调 Rab7 激活、内体 LDL 运输和 NPC1 依赖性溶酶体胆固醇输出方面，在细胞胆固醇稳态中发挥着核心作用。
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