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: 8463209
• 负责人: TODD R GRAHAM
• 金额: $ 30.81万
• 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 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463209
• 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; Transcription Factor AP-1; Transport Vesicles; Vesicle; Wound Healing; attenuation; base; cardiovascular disorder prevention; extracellular; in vitro Assay; male; member; mutant; novel; oxysterol binding protein; phosphatidylinositol 4-phosphate; preference; protein transport; proteoliposomes; public health relevance; reconstitution; tool; trans-Golgi Network; translocase; 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; Transcription Factor AP-1; Transport Vesicles; Vesicle; Wound Healing; attenuation; base; cardiovascular disorder prevention; extracellular; in vitro Assay; male; member; mutant; novel; oxysterol binding protein; 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.

描述（由申请人提供）：IV型P型ATPases（P4-ATPases）是一个大型推定的磷脂易位酶，或Flippases，涉及生物膜中磷脂不对称的生成和维持。人们认为，P4-ATPase将特异性脂质底物（例如磷脂酰甲酯（PS）和磷脂酰乙醇胺（PE））从细胞外叶片到膜的细胞叶片产生不对称性。在血细胞中最好理解不对称质膜的医学意义，在血液细胞中，调节磷脂酰丝氨酸（PS）在细胞外叶片上的暴露会诱导血液凝结。此外，经历了程序性细胞死亡的细胞还暴露于细胞外小叶上的PS，从而促进了其他细胞的识别和吞噬作用。因此，膜磷脂不对称的正常建立和调节在预防心血管疾病和在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P4-ATPase（ATP8B1）的缺乏会导致家族性肝内胆汁淤积，这种疾病是胆管膜中PS不对称性的损失导致分泌的胆汁损害该膜，最终导致肝衰竭。小鼠的P4-ATPase缺乏与饮食诱导的肥胖症和2型糖尿病（ATP10A和ATP10D）以及男性生育能力降低有关（ATP8B3）。在酿酒酵母的出现酵母糖酵母中的P4-ATP酶（DRS2，NEO1，DNF1，DNF2和DNF3）允许应用强大的分子遗传工具来解剖这些潜在弹药的生物化学和细胞生物学功能。除了支持产生膜不对称性的拟议功能外，这些研究令人惊讶地表明，在分泌和内吞途径中，囊泡介导的蛋白转运需要P4-ATPases。 DRS2定位于反式高尔基网络（TGN），是通过一种独立于网状蛋白外套募集到膜的机制来从该细胞器中从该细胞器中芽的AP-1/胶蛋白涂层的囊泡。假设DRS2直接将磷脂底物泵送到整个TGN膜上，以诱导膜曲率，该膜曲率被网状蛋白捕获并成型为囊泡。提出的研究将首次确定P4-ATPase（DRS2）是否足够以纯化的形式足以直接催化蛋白质脂质体中的磷脂氟脂脂活性。天然底物偏好将在重构系统中确定，以及非催化亚基（CDC50）对Flippase活性的贡献。将测试DRS2活性对膜曲率和囊泡形成与蛋白脂质体和分离的TGN膜的影响。此外，初步研究表明，DRS2是控制TGN（磷脂酰肌醇4-磷酸，ARFGEF，KES1）的重要分子的新型效应子，并且将确定该调节的机械基础。