使用体外重组试验测量胆固醇从溶酶体到过氧化物酶体的转移

Measurement of Cholesterol Transfer from Lysosome to Peroxisome Using an In Vitro Reconstitution Assay.

作者信息

Luo Jie, Liao Ya-Cheng, Xiao Jian, Song Bao-Liang

机构信息

Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 430072, China.

State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.

出版信息

Methods Mol Biol. 2017;1583:141-161. doi: 10.1007/978-1-4939-6875-6_11.

DOI:10.1007/978-1-4939-6875-6_11

PMID:28205172

Abstract

Low-density lipoproteins (LDLs) are taken up by the cell mainly through receptor-mediated endocytosis. LDL-derived cholesterol leaves lysosome and further transports to downstream organelles for specific cellular needs. We recently report that cholesterol transfers from lysosome to peroxisome through lysosome-peroxisome membrane contact (LPMC). Here, we use iodixanol density gradient centrifugation to isolate lysosomes and peroxisomes separately for the in vitro reconstitution of LPMC. We also apply H-cholesterol-labeled lysosomes and peroxisomes in vitro to measure H-cholesterol transfer through LPMC.

摘要

低密度脂蛋白（LDLs）主要通过受体介导的内吞作用被细胞摄取。LDL衍生的胆固醇离开溶酶体，并进一步转运至下游细胞器以满足特定的细胞需求。我们最近报道，胆固醇通过溶酶体-过氧化物酶体膜接触（LPMC）从溶酶体转移至过氧化物酶体。在此，我们使用碘克沙醇密度梯度离心法分别分离溶酶体和过氧化物酶体，用于LPMC的体外重建。我们还在体外应用H-胆固醇标记的溶酶体和过氧化物酶体来测量通过LPMC的H-胆固醇转移。