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非常薄的膜。该细胞膜或质膜具有脂质组分和蛋白质组分，所述脂质组分为极性或带电分子的通过提供屏障，所述蛋白质组分允许特权分子通过以使膜选择性地可渗透。磷脂是质膜的关键组成部分，这些两亲分子并排包装在一起形成双层片。每个小叶中的极性磷脂头基面向外以与水相互作用，并且疏水性脂肪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，这种现象称为“膜不对称”。面向哺乳动物细胞胞质溶胶的内小叶富含磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE），而细胞外小叶富含鞘脂、鞘糖脂和磷脂酰胆碱（PC）。 至少两类主动转运蛋白能够使磷脂穿过膜双层以建立膜不对称性，包括IV型P型ATP酶（P4-ATP酶）和ATP结合盒（ABC）转运蛋白家族的成员。令人惊讶的是，即使经过数十年的研究和一些ABC转运蛋白的X射线晶体结构，脂质如何转运仍然是一个谜。对于离子转运P型ATP酶（I、II和III型），X射线晶体结构也已得到解决，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酶是否进化出识别这个典型位点中更大的磷脂底物的能力，或者进化出独特的转运机制一直是争论的主题。初步数据支持这一建议强烈表明，P4-ATP酶是使用一个非经典的运输途径，翻转其磷脂底物。我们建议，磷脂被选择在细胞外小叶附近的“入口门”和细胞溶质小叶附近的“出口门”。该项目的研究将通过突变研究来验证这一双门假说，并更好地定义P4-ATP酶的磷脂转运机制。所获得的信息将有助于我们了解人类疾病是如何从膜不对称或P4-ATP酶缺陷中产生的。

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.