Drs2p Function in Clathrin-coated Vesicle Budding

描述(由申请人提供)：IV型P型ATPase(P4-ATPase)是一个推测的磷脂转位酶或Flppase的大家族，与生物膜中磷脂不对称性的产生和维持有关。P4-ATPase被认为是直接将特定的脂质底物，如磷脂酰丝氨酸(PS)和磷脂酰乙醇胺(PE)，从细胞膜的胞外小叶输送到胞内小叶，从而产生不对称性。不对称质膜的医学意义在血细胞中被最好地理解，在血细胞中，磷脂酰丝氨酸(PS)被调节地暴露在细胞外小叶上诱导血液凝结。此外，经历细胞程序性死亡的细胞也暴露在细胞外小叶上的PS，促进它们被其他细胞识别和吞噬。因此，膜磷脂不对称性的正常建立和调节在心血管疾病的预防以及发育和创伤修复过程中的组织重塑中起着至关重要的作用。此外，人类P4-ATPase(Atp8b1)的缺陷会导致家族性肝内胆汁淤积症，这种疾病的胆小管膜PS不对称性的丧失会导致分泌的胆汁破坏这一膜，最终导致肝功能衰竭。小鼠的P4-ATPase缺陷与饮食引起的肥胖和2型糖尿病(Atp10a和Atp10d)以及男性生育力下降(Atp8b3)有关。酿酒酵母芽生菌株(Drs2、Neo1、Dnf1、Dnf2和Dnf3)中P4-ATPase的特性使我们能够应用强大的分子遗传学工具来剖析这些潜在的Flippase的生化和细胞生物学功能。除了支持P4-ATPase在膜不对称形成中的作用外，这些研究还令人惊讶地表明，P4-ATPase是囊泡介导的蛋白质在分泌和内吞途径中运输所必需的。Drs2定位于反式高尔基体网络(TGN)，并需要通过一种独立于膜上网织蛋白涂层的机制从该细胞器中产生AP-1/网状蛋白包裹的囊泡。假设Drs2直接将磷脂底物泵到TGN膜上，诱导膜弯曲，膜曲率被网状蛋白捕获并模塑成囊泡。这项拟议的研究将首次确定P4-ATPase(DRS2)的纯化形式是否足以直接催化蛋白质脂质体中的磷脂翻转酶活性。在重组体系中，天然底物的偏好以及非催化亚基(CDC50)对翻转酶活性的贡献将被确定。将测试DRS2活性对蛋白脂质体和分离的TGN膜的膜曲率和囊泡形成的影响。此外，初步研究表明，Drs2是一种新的调控TGN(磷脂酰肌醇4-磷酸)囊泡萌发的重要分子的效应者，这一调控的机制基础将被确定。 公共卫生相关性：IV型P型ATPase(P4-ATPase)缺陷与肝病、肥胖和II型糖尿病有关。尽管越来越多的证据表明这些泵是控制膜磷脂不对称和囊泡介导的蛋白质运输的磷脂翻转酶，但P4-ATPase的确切生化和细胞生物学功能仍不确定。这项拟议的研究将确定P4-ATPase是否催化磷脂翻转酶的活性，并将确定P4-ATPase活性如何与高尔基复合体中笼罩着的囊泡的萌发相耦合的分子机制。

DESCRIPTION (provided by applicant): Type IV P-type ATPases (P4-ATPases) are a large family of putative phospholipid translocases, or flippases, implicated in the generation and maintenance of phospholipid asymmetry in biological membranes. It is thought that P4-ATPases directly pump specific lipid substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the extracellular leaflet to the cytosolic leaflet of a membrane to produce asymmetry. The medical significance of an asymmetric plasma membrane is best understood in blood cells where regulated exposure of phosphatidylserine (PS) on the extracellular leaflet induces blood clotting. In addition, cells undergoing programmed cell death also expose PS on the extracellular leaflet facilitating their recognition and phagocytosis by other cells. Thus, normal establishment and regulation of membrane phospholipid asymmetry plays a critical role in prevention of cardiovascular disease and in tissue remodeling during development and wound repair. Moreover, deficiency of a human P4-ATPase (Atp8b1) causes familial intrahepatic cholestasis, a disease where loss of PS asymmetry in the bile canalicular membrane leads to damage of this membrane by secreted bile, ultimately leading to liver failure. P4-ATPase deficiency in mice is linked to diet induced obesity and type 2 diabetes (Atp10a and Atp10d) as well as decreased male fertility (Atp8b3). Characterization of P4-ATPases in the budding yeast Saccharomyces cerevisiae (Drs2, Neo1, Dnf1, Dnf2 and Dnf3) has allowed the application of powerful molecular genetic tools to dissect the biochemical and cell biological functions of these potential flippases. In addition to supporting the proposed function in generating membrane asymmetry, these studies have surprisingly shown that P4-ATPases are required for vesicle-mediated protein transport in the secretory and endocytic pathways. Drs2 localizes to the trans-Golgi network (TGN) and is required to bud AP-1/clathrin-coated vesicles from this organelle by a mechanism that is independent of clathrin coat recruitment to the membrane. It is hypothesized that Drs2 directly pumps phospholipid substrates across the TGN membrane to induce membrane curvature that is captured and molded by clathrin into a vesicle. The proposed studies will determine for the first time if a P4-ATPase (Drs2) is sufficient in a purified form to directly catalyze phospholipid flippase activity in proteoliposomes. The native substrate preference will be determined in the reconstituted system as well as the contribution of the noncatalytic subunit (Cdc50) to flippase activity. The influence of Drs2 activity on membrane curvature and vesicle formation with proteoliposomes and isolated TGN membranes will be tested. In addition, preliminary studies indicate that Drs2 is a novel effector of important molecules controlling vesicle budding from the TGN (phosphatidylinositol 4-phosphate, ArfGEF, Kes1) and the mechanistic basis for this regulation will be determined. PUBLIC HEALTH RELEVANCE: Deficiencies in type IV P-type ATPases (P4-ATPases) are linked to liver disease, obesity and type II diabetes. The precise biochemical and cell biological function of P4-ATPases is still uncertain, although a growing body of evidence suggests that these pumps are phospholipid flippases that control membrane phospholipid asymmetry and vesicle- mediated protein transport. The proposed studies will determine if a P4-ATPase catalyzes phospholipid flippase activity and will define the molecular mechanisms for how a P4- ATPase activity is coupled to the budding of clathrin-coated vesicles from the Golgi complex.