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.

中文翻译：

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GARP复合物涉及通过靶向NPC2到溶酶体的细胞内胆固醇运输。

适当的细胞内胆固醇运输对细胞功能至关重要。两种溶酶体驻留蛋白NPC1和NPC2介导低密度脂蛋白衍生的胆固醇从溶酶体中流出。但是，与该过程有关的其他蛋白质在很大程度上仍然未知。通过基于两性霉素B的选择，我们分离了两种胆固醇转运缺陷型细胞系。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）复杂的其他亚基的消耗会损害NPC2到溶酶体的分选，并导致胆固醇积聚。GARP的缺乏阻碍了阳离子非依赖性甘露糖6磷酸受体（CI-MPR）向反高尔基网络的检索。进一步，Vps54突变小鼠显示出降低的细胞NPC2蛋白水平和增加的胆固醇积累，强调了GARP复合物在胆固醇转运中的生理作用。我们得出的结论是，GARP复合物通过以CI-MPR依赖性方式将NPC2靶向溶酶体，从而促进了细胞内胆固醇的运输。