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Drs2p Function in Clathrin-coated Vesicle Budding

Drs2p 在网格蛋白包被的囊泡出芽中的功能

基本信息

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
项目类别：
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

项目摘要

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ATP酶（P4-ATP酶）是一个大家族的推定磷脂移位酶或翻转酶，涉及生物膜中磷脂不对称性的产生和维持。据认为，P4-ATP酶直接泵特定的脂质底物，如磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE），从细胞外小叶到细胞溶质小叶的膜，以产生不对称性。不对称质膜的医学意义在血细胞中得到最好的理解，其中细胞外小叶上磷脂酰丝氨酸（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暴露于细胞外小叶上的PS，从而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预防心血管疾病和在发育和创伤修复期间的组织重塑中起关键作用。此外，人P4-ATP酶（Atp 8b 1）的缺乏导致家族性肝内胆汁淤积，这是一种其中胆小管膜中PS不对称性的丧失导致分泌的胆汁对该膜的损伤，最终导致肝功能衰竭的疾病。小鼠中的P4-ATP酶缺乏与饮食诱导的肥胖和2型糖尿病（Atp 10a和Atp 10 d）以及雄性生育力降低（Atp 8b 3）有关。芽殖酵母酿酒酵母（Saccharomycescerevisiae）中P4-ATP酶（Drs 2、Neo 1、Dnf 1、Dnf 2和Dnf 3）的特性使得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性的功能之外，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转运所必需的。Drs 2定位于trans-Golgi网络（TGN），并需要芽AP-1/网格蛋白包被的囊泡从这个细胞器的机制是独立的网格蛋白涂层招聘到膜。假设Drs 2直接泵送磷脂底物穿过TGN膜以诱导被网格蛋白捕获并模塑成囊泡的膜弯曲。拟议的研究将首次确定P4-ATP酶（Drs 2）是否足以以纯化形式直接催化脂蛋白体中的磷脂翻转酶活性。将在重构系统中确定天然底物偏好以及非催化亚基（Cdc 50）对翻转酶活性的贡献。将测试Drs 2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，Drs 2是一种新的效应器的重要分子控制囊泡出芽从TGN（磷脂酰肌醇4-磷酸，ArfGEF，Kes 1）和这种调节的机制基础将被确定。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Regulation of a Golgi flippase by phosphoinositides and an ArfGEF.

DOI：
10.1038/ncb1989
发表时间：
2009-12
期刊：
NATURE CELL BIOLOGY
影响因子：
21.3
作者：
Natarajan, Paramasivam;Liu, Ke;Patil, Dustin V.;Sciorra, Vicki A.;Jackson, Catherine L.;Graham, Todd R.
通讯作者：
Graham, Todd R.

Interplay of proteins and lipids in generating membrane curvature.