Multivesicular Body Formation Requires OSBP-Related Proteins and Cholesterol
Multivesicular Body Formation Requires OSBP-Related Proteins and Cholesterol
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DOI：
10.1371/journal.pgen.1001055
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发表时间：
2010-08-01
期刊：
影响因子：
4.5
通讯作者：
Arai, Hiroyuki
中科院分区：
文献类型：
--
作者：
Kobuna, Hiroyuki;Inoue, Takao;Arai, Hiroyuki
在真核生物中，不同的亚细胞器具有不同的胆固醇浓度，这被认为是生物功能的关键。氧化固醇结合蛋白相关蛋白（ORP）已被假定为介导细胞中的非囊泡胆固醇运输;然而，它们的体内功能以及胆固醇在每个细胞器中的生物学意义尚未完全了解。在这里，通过在秀丽隐杆线虫中产生ORPs的缺失突变体，我们表明ORPs是多泡体（MVB）的形成和功能所必需的。在使用obr四重突变体（obr-1; -2; -3; -4）的RNAi增强子筛选中，我们发现MVB相关基因与obr基因显示出强的遗传相互作用。在obr四重突变体中，晚期内体/溶酶体扩大，膜蛋白降解延迟，尽管内吞的可溶性蛋白通常被递送到溶酶体并降解。我们还发现，在突变体中，晚期内体/溶酶体的胆固醇含量降低。在野生型蠕虫中，胆固醇限制诱导形成扩大的晚期内体/溶酶体，如在obr四重突变体中观察到的，并且在MVB相关基因敲低后增加胚胎致死率。最后，我们表明，敲低的ORP 1 L，哺乳动物的ORP家族成员，诱导形成扩大的MVBs在HeLa细胞。我们的体内研究结果表明，正常的MVB形成和MVB介导的膜蛋白降解需要由ORP产生的晚期内体/溶酶体的适当胆固醇水平。
In eukaryotes, different subcellular organelles have distinct cholesterol concentrations, which is thought to be critical for biological functions. Oxysterol-binding protein-related proteins (ORPs) have been assumed to mediate nonvesicular cholesterol trafficking in cells; however, their in vivo functions and therefore the biological significance of cholesterol in each organelle are not fully understood. Here, by generating deletion mutants of ORPs in Caenorhabditis elegans, we show that ORPs are required for the formation and function of multivesicular bodies (MVBs). In an RNAi enhancer screen using obr quadruple mutants (obr-1; -2; -3; -4), we found that MVB-related genes show strong genetic interactions with the obr genes. In obr quadruple mutants, late endosomes/lysosomes are enlarged and membrane protein degradation is retarded, although endocytosed soluble proteins are normally delivered to lysosomes and degraded. We also found that the cholesterol content of late endosomes/lysosomes is reduced in the mutants. In wild-type worms, cholesterol restriction induces the formation of enlarged late endosomes/lysosomes, as observed in obr quadruple mutants, and increases embryonic lethality upon knockdown of MVB-related genes. Finally, we show that knockdown of ORP1L, a mammalian ORP family member, induces the formation of enlarged MVBs in HeLa cells. Our in vivo findings suggest that the proper cholesterol level of late endosomes/lysosomes generated by ORPs is required for normal MVB formation and MVB-mediated membrane protein degradation.