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Phospholipid Flip-flop in Biogenic Membranes

生物膜中的磷脂触发器

基本信息

批准号：
8389633
负责人：
ANANT K MENON
金额：
$ 48.85万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8389633
关键词：
ATP Hydrolysis ATP phosphohydrolase ATP-Binding Cassette Transporters Abbreviations Acetylcholinesterase Active Biological Transport Affinity Anabolism Antibodies B cell differentiation B-Lymphocytes Bacteria Biochemical Biochemical Genetics Biological Carrier Proteins Cell Wall Cell membrane Cell surface Collection Concanavalin A Data Diffusion Dissection Dolichol Dolichol Phosphates Embryo Endoplasmic Reticulum Escherichia coli Eukaryota Face Family Foundations Genetic Glucose Glycerophospholipids Glycoconjugates Glycolipids Glycoproteins Glycosphingolipids Glycosylphosphatidylinositols Goals Head Human In Vitro Lateral Lecithin Link Lipids Lipopolysaccharides Liver Mammals Mannose Mass Spectrum Analysis Membrane Membrane Lipids Membrane Proteins Microbial Physiology Molecular Neural Cell Adhesion Molecules O Antigens Oligosaccharides Organelles Phospholipids Plasma Play Polysaccharides Positioning Attribute Post-Translational Protein Processing Preparation Protein Glycosylation Protein Precursors Proteins Publishing Rattus Reaction Research Role Side Specificity System Thin Layer Chromatography Time Triton Tritons Work Yeasts cell growth choleragen receptor dolichol pyrophosphate glycosylation in vivo interest lipid transport man mannose-phosphate-citronellol mannosyl(5)-N-acetyl(2)-glucose membrane biogenesis novel protein purification public health relevance reconstitution screening

项目摘要

DESCRIPTION (provided by applicant): The bi-directional translocation of lipids from one side of a biological membrane to the other is termed flip-flop. Lipid flip-flop across the endoplasmic reticulum (ER) membrane is required for protein N-glycosylation and GPI-anchoring. These protein modifications are essential in eukaryotes; for example, their genetic abrogation causes embryonic lethality in mammals and renders yeast unviable. Lipid flip-flop across the ER is also required for membrane biogenesis: phospholipids that are synthesized on the cytoplasmic face of the ER must be translocated to the opposite face to enable the membrane bilayer to grow uniformly. The demand for lipid flip-flop at the ER is likely to be exceptionally high when the ER membrane expands and glycoprotein secretion increases; this occurs, for example, during the differentiation of B-lymphocytes to antibody-secreting plasma B cells. Unassisted flip-flop is extremely slow because of the energy barrier to taking the polar lipid head group through the hydrophobic interior of the membrane, yet lipids flip-flop rapidly across the ER membrane on a time-scale of seconds. This is because the ER possesses specific transport proteins (flippases) that accelerate lipid flipping to a physiologically sufficient rate. Lipid flipping in the ER occurs by an ATP-independent mechanism in which the flippases facilitate 'downhill' transport of lipids; this distinguishes ER flippases from other translocators, typically found in the eukaryotic plasma membrane, that couple ATP hydrolysis to concentrative 'uphill' transport of lipids. We estimate that there are as many as six different ER lipid flippases but none of these have been identified at the molecular level. We developed biochemical reconstitution systems that recapitulate the activity of three of the flippases required for ER membrane bilayer expansion and protein glycosylation. These flippases specifically translocate glycerophospholipids, oligosaccharide diphosphate dolichols and mannose-phosphate dolichol. Our aim is to identify these physiologically important translocators with the long-term goal of understanding their mechanism of action. We propose to do this via a two-pronged approach involving protein purification and mass spectrometry on the one hand, and screening of systematic collections of yeast ER membrane proteins on the other. Our purification efforts will be aided by the use of novel affinity matrices. We will also use partially purified flippase preparations to continue our efforts to define the specificity of these proteins. Our published work and preliminary data put us in an excellent position to accomplish these aims.

描述(由申请人提供)：脂质从生物膜的一侧到另一侧的双向转移称为触发器。蛋白质N-糖基化和GPI锚定需要跨过内质网(ER)膜的脂质翻转。这些蛋白质修饰在真核生物中是必不可少的；例如，它们的基因缺失会导致哺乳动物的胚胎死亡，并使酵母无法存活。膜生物发生也需要跨越内质网的脂质触发：在内质网细胞质表面合成的磷脂必须转移到相反的表面，使膜双层能够均匀生长。当ER膜扩张和糖蛋白分泌增加时，内质网对脂质触发的需求可能会异常高；例如，在B淋巴细胞分化为分泌抗体的血浆B细胞的过程中。无辅助触发是极其缓慢的，因为带着极性脂头基团通过膜的疏水内部的能量障碍，然而脂类以秒为时间尺度快速地穿过内质网膜。这是因为内质网具有特定的转运蛋白(翻转酶)，可以加速脂肪翻转到生理上足够的速度。内质网中的脂质翻转是通过一种不依赖于ATP的机制发生的，在这种机制中，翻转酶促进了脂类的下坡运输；这将ER翻转酶与其他转运子区分开来，这些转运子通常存在于真核细胞的质膜中，将ATP水解与脂质的集中“上行”运输结合在一起。我们估计有多达六种不同的内质网脂质翻转酶，但没有一个在分子水平上被鉴定出来。我们开发了生化重建系统，概括了内质网膜双层扩张和蛋白质糖基化所需的三种翻转酶的活性。这些翻转酶能特异性地转运甘油磷脂、寡糖二磷酸多糖醇和甘露糖磷酸多糖醇。我们的目标是识别这些具有重要生理意义的转运体，长期目标是了解它们的作用机制。我们建议通过双管齐下的方法做到这一点，一方面涉及蛋白质纯化和质谱学，另一方面筛选酵母ER膜蛋白的系统集合。我们的纯化工作将通过使用新的亲和矩阵而得到帮助。我们还将使用部分纯化的Flipase制剂来继续我们的努力，以确定这些蛋白质的特异性。我们发表的工作和初步数据使我们处于实现这些目标的有利地位。