Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop
Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop
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DOI：
10.1016/j.bpj.2016.05.023
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发表时间：
2016-06-21
影响因子：
3.4
通讯作者：
Nakano, Minoru
中科院分区：
文献类型：
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作者：
Nakao, Hiroyuki;Ikeda, Keisuke;Nakano, Minoru
磷脂在内质网（ER）膜中快速翻转的机制仍然未知。我们先前证明，模型跨膜肽序列中心的亲水性残基的存在有效地促进了磷脂翻转，并且亲水性残基占SOSUI软件预测的人ER膜蛋白跨膜序列中心区域的4.5%。我们推测，ER蛋白与亲水性残基可能发挥关键作用，促进翻转。在这里，我们评估了翻转率的荧光标记的脂质在囊泡含有每一个11个合成肽的跨膜序列，使用连二亚硫酸盐淬灭测定。虽然翻转酶活性的9个肽出乎意料地低，基于EDEM 1和SPAST蛋白质的肽显示出增强的翻转酶活性与三种不同的荧光标记的脂质。在EDEM 1或SPAST肽的中心区域分别用His或Arg取代疏水性Ala，减弱了它们翻转磷脂的能力。有趣的是，在EDEM 1或SPAST中心区域以外的位置分别用Arg或His取代Ala也会影响触发器的增强。这些结果表明，Arg和His对于这两种肽增加翻转速率的能力都是重要的。EDEM 1肽在显著低的肽浓度下表现出高活性，表明Arg和His在α-螺旋结构中的同侧定位对于翻转促进是关键的，并且EDEM 1蛋白是ER中的候选翻转酶。
The mechanism whereby phospholipids rapidly flip-flop in the endoplasmic reticulum (ER) membrane remains unknown. We previously demonstrated that the presence of a hydrophilic residue in the center of the model transmembrane peptide sequence effectively promoted phospholipid flip-flop and that hydrophilic residues composed 4.5% of the central regions of the membrane-spanning sequences of human ER membrane proteins predicted by SOSUI software. We hypothesized that ER proteins with hydrophilic residues might play a critical role in promoting flip-flop. Here, we evaluated the flip rate of fluorescently labeled lipids in vesicles containing each of the 11 synthetic peptides of membrane-spanning sequences, using a dithionitequenching assay. Although the flippase activities of nine peptides were unexpectedly low, the peptides based on the EDEM1 and SPAST proteins showed enhanced flippase activity with three different fluorescently labeled lipids. The substitution of hydrophobic Ala with His or Arg in the central region of the EDEM1 or SPAST peptides, respectively, attenuated their ability to flip phospholipids. Interestingly, substituting Ala with Arg or His at a location outside of the central region of EDEM1 or SPAST, respectively, also affected the enhancement of flip-flop. These results indicated that both Arg and His are important for the ability of these two peptides to increase the flip rates. The EDEM1 peptide exhibited high activity at significantly low peptide concentrations, suggesting that the same side positioning of Arg and His in alpha-helix structure is critical for the flip-flop promotion and that the EDEM1 protein is a candidate flippase in the ER.