Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China.
The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Cell Rep. 2017 Jun 27;19(13):2823-2835. doi: 10.1016/j.celrep.2017.06.012.
Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner.
适当的细胞内胆固醇转运对于细胞功能至关重要。两种溶酶体驻留蛋白 NPC1 和 NPC2 介导从溶酶体中排出 LDL 衍生的胆固醇。然而，该过程中涉及的其他蛋白质在很大程度上仍不清楚。通过两性霉素 B 为基础的选择，我们分离出两种胆固醇转运缺陷的细胞系。随后的全转录组测序分析显示，两个细胞系在液泡蛋白分选 53（Vps53）基因中具有相同的突变。VPS53 或高尔基体相关逆行蛋白（GARP）复合物的其他亚基的耗竭会损害 NPC2 向溶酶体的分拣，并导致胆固醇积累。GARP 缺陷会阻止阳离子非依赖性甘露糖 6-磷酸受体（CI-MPR）向反式高尔基网络的回收。此外，Vps54 突变小鼠显示细胞 NPC2 蛋白水平降低和胆固醇积累增加，强调了 GARP 复合物在胆固醇转运中的生理作用。我们的结论是，GARP 复合物通过以 CI-MPR 依赖的方式将 NPC2 靶向溶酶体，从而有助于细胞内胆固醇转运。
