Drs2p Function in Clathrin-coated Vesicle Budding

Drs2p 在网格蛋白包被的囊泡出芽中的功能

• 批准号: 7781106
• 负责人: TODD R GRAHAM
• 金额: $ 30.99万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7781106
• 关键词: ATP phosphohydrolase; Apoptosis; Bile fluid; Binding; Biochemical; Biological; Biological Assay; Biological Process; Blood Cells; Blood Clot; Blood coagulation; C-terminal; Cell membrane; Cells; Clathrin; Clathrin-Coated Vesicles; Complex; Coupled; Defect; Development; Diet; Disease; Early Endosome; Family; Fertility; Funding; Generations; Goals; Golgi Apparatus; Human; Link; Lipids; Liposomes; Liver Failure; Liver diseases; Maintenance; Mediating; Medical; Membrane; Molds; Molecular; Molecular Chaperones; Molecular Genetics; Multivesicular Body; Mus; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organelles; Pathway interactions; Phagocytosis; Phenocopy; Phosphatidylethanolamine; Phosphatidylserines; Phospholipids; Play; Progressive intrahepatic cholestasis; Proteins; Pump; Regulation; Role; Saccharomyces cerevisiae; Saccharomycetales; System; Tail; Testing; Time; Tissues; Transport Vesicles; Vesicle; Wound Healing; attenuation; base; cardiovascular disorder prevention; extracellular; in vitro Assay; male; member; mutant; novel; oxysterol binding protein; phosphatidylethanolamine; phosphatidylinositol 4-phosphate; preference; protein transport; proteoliposomes; public health relevance; reconstitution; tool; trans-Golgi Network; translocase

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.

描述（由申请人提供）：IV型p型atp酶（p4 - atp酶）是一大家族的磷脂转位酶或翻转酶，与生物膜中磷脂不对称的产生和维持有关。据认为，p4 - atp酶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），从细胞外小叶泵送到膜的胞质小叶，从而产生不对称。不对称质膜的医学意义在血细胞中得到了最好的理解，其中细胞外小叶上的磷脂酰丝氨酸（PS）的调节暴露诱导血液凝固。此外，经历程序性细胞死亡的细胞也会将PS暴露在细胞外小叶上，以促进其他细胞对其的识别和吞噬。因此，细胞膜磷脂不对称的正常建立和调控在心血管疾病的预防以及发育和伤口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 - atp酶（Atp8b1）的缺乏会导致家族性肝内胆汁淤积症，这种疾病是指胆小管膜PS不对称的丧失导致该膜被分泌的胆汁破坏，最终导致肝衰竭。小鼠p4 - atp酶缺乏与饮食引起的肥胖和2型糖尿病（Atp10a和Atp10d）以及男性生育能力下降（Atp8b3）有关。对酿酒酵母p4 - atp酶（Drs2、Neo1、Dnf1、Dnf2和Dnf3）的研究，使我们能够应用强大的分子遗传学工具来剖析这些潜在翻转酶的生化和细胞生物学功能。除了支持所提出的产生膜不对称的功能外，这些研究还令人惊讶地表明，p4 - atp酶是分泌和内吞途径中囊泡介导的蛋白质运输所必需的。Drs2定位于反式高尔基网络（TGN），并需要通过一种独立于网格蛋白外壳向膜募集的机制从该细胞器中产生AP-1/网格蛋白包被囊泡。据推测，Drs2直接泵送磷脂底物穿过TGN膜，诱导被网格蛋白捕获并塑造成囊泡的膜曲率。拟议的研究将首次确定p4 - atp酶（Drs2）是否足以在纯化形式下直接催化蛋白脂质体中的磷脂翻转酶活性。天然底物的偏好将在重组体系中确定，以及非催化亚基（Cdc50）对翻转酶活性的贡献。将测试Drs2活性对膜曲率和蛋白脂质体及分离TGN膜囊泡形成的影响。此外，初步研究表明Drs2是调控TGN囊泡出芽的重要分子（phosphatidylinositol 4-phosphate, ArfGEF, Kes1）的新效应分子，并将确定其调控的机制基础。