Phospholipid Flip-flop in Biogenic Membranes

生物膜中的磷脂触发器

• 批准号: 8197567
• 负责人: ANANT K MENON
• 金额: $ 35.41万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197567
• 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; Screening procedure; 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

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. PUBLIC HEALTH RELEVANCE: Flipping of lipids from one side of a biological membrane to the other is necessary for membrane expansion during cell growth, as well as for the biosynthesis of molecules that play critical roles in human and microbial physiology. These molecules include glycoproteins such as the neural cell adhesion molecule, GPI-anchored proteins such as acetylcholinesterase, glycolipids such as the receptor for cholera toxin, components of the cell walls of bacteria and yeast, and the O-antigen of E. coli lipopolysaccharide. We are interested in identifying the transport proteins that catalyze lipid flipping in yeast and mammals and understanding how they work.

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粘结蛋白需要跨内质网（ER）膜的脂质触发器。这些蛋白质修饰在真核生物中至关重要。例如，它们的遗传废除会导致哺乳动物中的胚胎致死性，并使酵母无法生存。膜生物发生也需要穿过ER的脂质触发器：在ER的细胞质面上合成的磷脂必须易位到相反的面，以使膜双层能够均匀生长。当ER膜扩展并增加糖蛋白分泌时，ER脂质触发器的需求可能异常高。例如，在B淋巴细胞与分泌抗体分泌血浆B细胞的分化过程中发生这种情况。 由于将极性脂质头部组穿过膜的疏水性内部的能量屏障，因此无助的触发器非常慢，但是脂质在几秒钟的时间尺度上迅速穿过ER膜。这是因为ER具有特定的转运蛋白（Flippase），该蛋白会加速脂质，从而将脂质变为生理上足够的速率。 ER中的脂质翻转是通过无关的机制发生的，其中Flippases促进了脂质的“下坡”转运。这将ER flippases与其他转运剂区分开，通常在真核质膜中发现，这对夫妇ATP水解为脂质的浓度“上坡”转运。 我们估计有多达六种不同的ER脂质氟脂酶，但在分子水平上都没有鉴定出来。我们开发了生化重构系统，这些系统概括了ER膜双层膨胀和蛋白质糖基化所需的三个Flippase的活性。这些Flippases专门将甘油磷脂，寡糖二磷酸二甲基和甘露糖 - 磷酸二甲醇分解。我们的目的是通过理解其作用机理的长期目标来识别这些重要性的转运剂。我们建议通过一方面涉及蛋白质纯化和质谱法的两种原始方法进行此操作，并筛选另一手的系统收集酵母ER膜蛋白。我们的纯化工作将通过使用新颖的亲和力矩阵来帮助。我们还将使用部分纯化的Flippase制剂来继续我们的努力来定义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绝佳位置。 公共卫生相关性：将脂质从生物膜的一侧翻转到另一侧，对于细胞生长过程中的膜扩张以及在人类和微生物生理中起关键作用的分子的生物合成是必要的。这些分子包括糖蛋白，例如神经细胞粘附分子，GPI锚定的蛋白，例如乙酰胆碱酯酶，糖脂，例如霍乱毒素的受体，细菌和酵母菌细胞壁的细胞壁成分，以及大肠杆菌的大肠杆菌。我们有兴趣确定催化脂质翻转酵母和哺乳动物的运输蛋白，并了解它们的工作原理。