Membrane-spanning peptides induce phospholipid flop:: A model for phospholipid translocation across the inner membrane of E-coli
Membrane-spanning peptides induce phospholipid flop:: A model for phospholipid translocation across the inner membrane of E-coli
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DOI：
10.1021/bi010627
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发表时间：
2001-09-04
期刊：
影响因子：
2.9
通讯作者：
de Kruijff, B
中科院分区：
文献类型：
--
作者：
Kol, MA;de Kroon, AIPM;de Kruijff, B
磷脂转运（翻牌）穿过大肠杆菌内膜的机制仍有待阐明。我们测试了这样的假设，即蛋白质的跨膜结构域仅仅通过它们在膜中的存在就催化了磷脂的翻转。作为模型，将氨基酸序列为GXXL(AL)(n)XXA （X = K， H或W， n = 8或12）的模拟蛋白质跨膜延伸的肽纳入大肠杆菌磷脂组成的大单层囊泡中。磷脂翻翻是通过测定2,6-（7-硝基2,1,3-苯并恶二唑-4-基）氨基丙基（C6NBD）标记的磷脂类似物对二亚硝酸盐的可及性的增加来测量的，最初只存在于囊泡膜的内小叶中。在加入GKKL(AL)(12)KKA的囊泡中观察到c6nbd -磷脂酰甘油（C6NBD-PG）的快速翻转，表观一级翻转速率常数（K-flop）随着肽与磷脂的摩尔比线性增加，在肽与磷脂的摩尔比为1:250时达到类似于10分钟的易位半衰期。GXXL(AL)(8)XXA系列多肽也能诱导C6NBD-PG的翻位，支持了蛋白质跨膜部分介导磷脂易位的假设。在这个系列中，K-flop按X = K > H > w的顺序递减，表明膜界面区域的肽-脂质相互作用调节了肽引起flop的效率。对于测试的肽，c6nbd -磷脂酰乙醇胺（C6NBD-PE）的翻转速度明显慢于C6NBD-PG。在没有肽的囊泡中，C6BD-PG和C6NBD-PE的翻牌都可以忽略不计。我们提出了一个考虑到观察到的肽和脂质特异性的肽诱导翻翻模型。
The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro2,1,3-benzoxadiazol-4-yl)aminocaproyl (C6NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C6NBD-phosphatidylglycerol (C6NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K-flop) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of similar to 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degreesC. The peptides of the series GXXL(AL)(8)XXA also induced flop Of C6NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K-flop decreased in the order X = K > H > W. indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop Of C6NBD-phosphatidylethanolamine (C6NBD-PE) was substantially slower than that of C6NBD-PG. In vesicles without peptide, flop was negligible both for C6BD-PG and for C6NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.