P4-ATPase mechanism of phospholipid translocation

描述（由申请人提供）：细胞的定义特征是区分细胞内和细胞外环境的令人难以置信的薄膜。这种细胞膜或质膜具有脂质成分，它为极性或带电分子的通过提供屏障，而蛋白质成分允许特权分子的通过，使膜具有选择性渗透性。磷脂是质膜的关键组成部分，这些两亲分子肩并肩地聚集在一起形成两层膜。每个小叶中的极性磷脂头基团面向外与水相互作用，疏水脂肪酸酰基链面向双层结构的内部。真核细胞膜的一个显著特征是这两层具有非常不同的磷脂组成，这种现象被称为“膜不对称”。哺乳动物细胞面向细胞质的内小叶富含磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE），细胞外小叶富含鞘脂、鞘脂糖和磷脂酰胆碱（PC）。至少有两类活性转运蛋白能够将磷脂穿过膜双分子层，从而建立膜不对称，包括IV型p型atp酶（p4 - atp酶）和atp结合盒转运蛋白家族成员。令人惊讶的是，即使经过几十年的研究和一些ABC转运蛋白的x射线晶体结构，脂质是如何运输的仍然是一个谜。使用离子传输的p型atp酶（I型，II型和III型），x射线晶体结构也得到了解决，突出了跨膜结构域中心的结构保守的底物结合位点（典型位点）。p4 - atp酶是否进化出了识别这个标准位点上更大的磷脂底物的能力，或者进化出了一种独特的运输机制，一直是争论的主题。支持这一建议的初步数据强烈表明，P4-ATPases使用非规范转运途径翻转其磷脂底物。我们认为磷脂在细胞外小叶附近的“入口门”和细胞质小叶附近的“出口门”都被选择。本项目的研究将通过突变研究验证这一双门假说，更好地明确P4- atp酶转运磷脂的机制。获得的信息将有助于我们了解人类疾病是如何由膜不对称缺陷或p4 - atp酶缺乏引起的。

DESCRIPTION (provided by applicant): The defining feature of a cell is the incredibly thin sheet of membrane that demarcates the intracellular and extracellular milieus. This cell membrane, or plasma membrane, has a lipid component that provides a barrier to passage of polar or charged molecules, and protein component that allows passage of privileged molecules to make the membrane selectively permeable. Phospholipids are critical building blocks of the plasma membrane and these amphipathic molecules pack together side-by-side to form a two-layered sheet. The polar phospholipid headgroups in each leaflet face outward to interact with water and the hydrophobic fatty acyl chains face the interior of the bilayer structure. A remarkable characteristic of the eukaryotic cell membrane is that these two layers have a very different phospholipid composition, a phenomenon known as "membrane asymmetry". The inner leaflet facing the cytosol of mammalian cells is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) while the extracellular leaflet is enriched in sphingolipids, glycosphingolipids and phosphatidylcholine (PC). At least two classes of active-transport proteins are capable of moving phospholipids across a membrane bilayer to establish membrane asymmetry, including the type IV P-type ATPases (P4-ATPases) and members of the ATP-binding cassette (ABC) transporter families. Amazingly, even with decades of study and x-ray crystal structures of some ABC transporters, how lipids are transported remains a mystery. With ion- transporting P-type ATPases (type I, II and III), x-ray crystal structure have also been solved highlighting a structurally conserved substrate binding site in the center of the transmembrane domain (a canonical site). Whether P4-ATPases evolved the ability to recognize the much larger phospholipid substrate in this canonical site, or evolved a unique transport mechanism has been the subject of debate. Preliminary data supporting this proposal strongly suggests that the P4-ATPases are using a noncanonical transport pathway to flip their phospholipid substrate. We propose that phospholipid is being selected at both an "entry gate" near the extracellular leaflet and an "exit gate" near the cytosolic leaflet. Studies in this projet will test this two-gate hypothesis through mutational studies and better define the mechanism of phospholipid transport by the P4- ATPases. The information obtained will help us understand how human diseases arise from defects in membrane asymmetry or P4-ATPase deficiency.