Use of mutant 125I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells
Use of mutant 125I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells
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DOI：
10.1073/pnas.1309273110
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发表时间：
2013-06-25
影响因子：
11.1
通讯作者：
Radhakrishnan, Arun
中科院分区：
文献类型：
--
作者：
Das, Akash;Goldstein, Joseph L.;Radhakrishnan, Arun
动物细胞严格控制胆固醇在其细胞器膜中的分布。这种调节需要一种有效的机制来在细胞器之间运输不溶性胆固醇。在本研究中，我们产生了一个I-125标记的突变体版本的穿孔素O（PFO），胆固醇结合蛋白，并使用它来测量完整细胞的质膜中的胆固醇。我们还从相同的细胞中纯化质膜，这使我们能够直接将胆固醇浓度与I-125-PFO结合联系起来。我们发现，胆固醇是有组织的质膜的方式，使其难以接近PFO，直到其浓度超过总脂质的35摩尔%的阈值。这个35%的阈值与以前发现的PFO与内质网膜结合的5%阈值形成鲜明对比。I-125-PFO探针也被证明可用于监测LDL衍生的胆固醇从溶酶体到质膜的移动。使用三种不同的突变体细胞系，我们表明，这种运输需要受体介导的摄取LDL，水解LDL-胆固醇酯在溶酶体中，并转移释放的胆固醇到质膜。
Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an I-125-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to I-125-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The I-125-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.