Role of caveolin-1 and cholesterol in transmembrane fatty acid movement
Role of caveolin-1 and cholesterol in transmembrane fatty acid movement
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DOI：
10.1021/bi051999b
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发表时间：
2006-03-07
期刊：
影响因子：
2.9
通讯作者：
Pilch, PF
中科院分区：
文献类型：
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作者：
Meshulam, T;Simard, JR;Pilch, PF
我们通过转染创建了一系列表达不同量小窝蛋白 - 1的HEK 293细胞系，以测试这种蛋白质在具有这种功能获得的细胞中对长链脂肪酸（FA）运输和代谢的可能影响。我们使用一种细胞外荧光探针（ADIFAB）来监测外源性FA与质膜的结合，并使用一种细胞内pH探针来监测FA在质膜两侧的平衡。实时荧光测量显示油酸快速结合到质膜的细胞外侧，并通过翻转机制快速跨脂质双层转运（<5秒）。两种表达的小窝蛋白 - 1水平与脂肪细胞大致相当（脂肪细胞具有非常高的内源性小窝蛋白 - 1表达水平）的细胞系，除了荧光的快速变化外，还显示细胞内pH的变化相对较慢（t(1/2)<100秒）。我们将这个额外的第二阶段解释为代表额外的FA从质膜的外层转运到内层。较慢的动力学可能代表FA在质膜高度有序、刚性区域的翻转较慢，或者FA与质膜内层的小窝蛋白 - 1结合。棕榈酸酯和反油酸酯（一种反式FA）跨膜运动的动力学与油酸酯相同。这些结果是在假定的FA转运蛋白CD36缺失以及脂肪酸转运蛋白（FATP）2和4的表达没有任何变化的情况下观察到的，并且与细胞游离胆固醇含量的增加直接相关。在所有细胞系中FA代谢都较慢，并且小窝蛋白 - 1的表达并没有增强它。我们得出结论，FA跨质膜的运输受小窝蛋白 - 1和胆固醇调节，并且不依赖于假定的FA转运蛋白CD36和FATP。
We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (< 5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.