Chemical modification identifies two populations of glycerophospholipid flippase in rat liver ER
Chemical modification identifies two populations of glycerophospholipid flippase in rat liver ER
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DOI：
10.1021/bi049063a
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发表时间：
2004-08-24
期刊：
影响因子：
2.9
通讯作者：
Menon, AK
中科院分区：
文献类型：
--
作者：
Chang, QL;Gummadi, SN;Menon, AK
内质网（ER）中甘油磷脂的跨双层翻转是膜生物发生的关键特征。翻转似乎是一种不依赖于 ATP 的双向过程，由特定蛋白质或翻转酶促进。尽管磷脂翻转酶尚未被鉴定，但最近通过生化重组研究获得了支持专用翻转酶存在的证据，表明去污剂溶解的内质网蛋白的某些色谱分离级分富含翻转酶活性，而其他部分则无活性。现在，我们通过描述利用荧光磷脂类似物作为转运报告基因的两种方便的翻转酶活性测定法来扩展这些研究。我们使用这些测定法来表明：(i) 由内质网富含翻转酶的 Triton X-100 提取物生成的蛋白脂质体可以翻转磷脂酰胆碱、磷脂酰乙醇胺和磷脂酰丝氨酸的类似物； (ii) 所有三种磷脂的翻转可能是由于相同的翻转酶而不是不同的磷脂特异性转运蛋白； (iii) 功能性翻转酶相当于 Triton 提取物中 1% (w/w) 的 ER 膜蛋白； (iv) ER 中的甘油磷脂翻转酶活性可归因于两种功能不同的蛋白质（或蛋白质类别），这两种蛋白质分别由它们对半胱氨酸和组氨酸修饰试剂 N-乙基马来酰亚胺和焦碳酸二乙酯的敏感性定义。对 N-乙基马来酰亚胺敏感类翻转酶活性的分析表明，翻转酶蛋白中功能关键的巯基埋藏在膜的疏水环境中，但在将蛋白提取到 Triton X-100 中时变得具有反应性。这一观察结果为未来通过亲和方法分离翻转酶的尝试带来了巨大的希望。
Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Flipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent similar to1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.