Objective

Cholesterol plays a pivotal role in mitochondrial steroidogenesis, membrane structure, and respiration. Mitochondrial membranes are intrinsically low in cholesterol content and therefore must be replenished with cholesterol from other subcellular membranes. However, the molecular mechanisms underlying mitochondrial cholesterol transport remains poorly understood. The Aster-B gene encodes a cholesterol binding protein recently implicated in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER). In this study, we investigated the function and underlying mechanism of Aster-B in mediating mitochondrial cholesterol transport.

Methods

CRISPR/Cas9 gene editing was carried out to generate cell lines deficient in Aster-B expression. The effect of Aster-B deficiency on mitochondrial cholesterol transport was examined by both confocal imaging analysis and biochemical assays. Deletion mutational analysis was also carried out to identify the function of a putative mitochondrial targeting sequence (MTS) at the N-terminus of Aster-B for its role in targeting Aster-B to mitochondria and in mediating mitochondrial cholesterol trafficking.

Results

Ablation of Aster-B impaired cholesterol transport from the ER to mitochondria, leading to a significant decrease in mitochondrial cholesterol content. Aster-B is also required for mitochondrial transport of fatty acids derived from hydrolysis of cholesterol esters. A putative MTS at the N-terminus of Aster-B mediates the mitochondrial cholesterol uptake. Deletion of the MTS or ablation of Arf1 GTPase which is required for mitochondrial translocation of ER proteins prevented mitochondrial cholesterol transport, leading to mitochondrial dysfunction.

Conclusions

We identified Aster-B as a key regulator of cholesterol transport from the ER to mitochondria. Aster-B also coordinates mitochondrial cholesterol trafficking with uptake of fatty acids derived from cholesterol esters, implicating the Aster-B protein as a novel regulator of steroidogenesis.

中文翻译：

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Aster-B 与 Arf1 协调以调节线粒体胆固醇转运。

客观的

胆固醇在线粒体类固醇生成、膜结构和呼吸中起关键作用。线粒体膜本身的胆固醇含量很低，因此必须补充来自其他亚细胞膜的胆固醇。然而，线粒体胆固醇转运的分子机制仍然知之甚少。Aster-B 基因编码最近涉及胆固醇从质膜运输到内质网 (ER) 的胆固醇结合蛋白。在这项研究中，我们研究了 Aster-B 在介导线粒体胆固醇转运中的功能和潜在机制。

方法

进行 CRISPR/Cas9 基因编辑以生成缺乏 Aster-B 表达的细胞系。通过共聚焦成像分析和生化测定检查了 Aster-B 缺乏对线粒体胆固醇转运的影响。还进行了缺失突变分析，以确定 Aster-B N 末端的假定线粒体靶向序列 (MTS) 在将 Aster-B 靶向线粒体和介导线粒体胆固醇运输中的作用。

结果

Aster-B 的消融损害了从 ER 到线粒体的胆固醇转运，导致线粒体胆固醇含量显着降低。Aster-B 也是胆固醇酯水解衍生的脂肪酸的线粒体转运所必需的。Aster-B N 末端的假定 MTS 介导线粒体胆固醇摄取。删除 MTS 或消除 ER 蛋白线粒体易位所需的 Arf1 GTPase 可阻止线粒体胆固醇转运，导致线粒体功能障碍。

结论

我们确定 Aster-B 是胆固醇从 ER 转运到线粒体的关键调节因子。Aster-B 还协调线粒体胆固醇运输与从胆固醇酯衍生的脂肪酸的摄取，暗示 Aster-B 蛋白是类固醇生成的新调节剂。