Iaea David B, Mao Shu, Lund Frederik W, Maxfield Frederick R
Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065.
Weill Cornell Medical College, Rockefeller University, and Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065.
Mol Biol Cell. 2017 Apr 15;28(8):1111-1122. doi: 10.1091/mbc.E16-07-0499. Epub 2017 Feb 16.
Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a =12-15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC.
胆固醇是哺乳动物细胞膜的重要组成成分。质膜和内吞循环区室（ERC）都富含胆固醇。细胞器间胆固醇的丰度和分布受到涉及囊泡和非囊泡甾醇转运过程的多种机制的严格控制。我们使用荧光胆固醇类似物脱氢麦角固醇，通过光漂白后的荧光恢复和一种新型甾醇流出测定法，研究了质膜和ERC之间的甾醇转运。我们发现，U2OS细胞系中这些细胞器间的甾醇转运半衰期为12 - 15分钟。大约70%的甾醇转运不依赖ATP，因此是非囊泡性的。细胞胆固醇水平的升高显著增加双向转运速率常数，但胆固醇水平降低仅有适度影响。一种可溶性甾醇转运蛋白STARD4，占质膜和ERC之间总甾醇转运的约25%以及非囊泡甾醇转运的约33%。这项研究表明，非囊泡甾醇转运机制，尤其是STARD4，在质膜和ERC之间的甾醇转运中占很大比例。
