Phospholipid Flip-flop in Biogenic Membranes

生物膜中的磷脂触发器

• 批准号: 7080488
• 负责人: ANANT K MENON
• 金额: $ 31.17万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7080488
• 关键词: animal tissue; endoplasmic reticulum; enzyme activity; membrane activity; membrane proteins; membrane structure; phospholipids; protein structure function; yeasts

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to elucidate the mechanism(s) by which polar lipids are flip-flopped across biogenic (self-synthesizing) membranes such as the endoplasmic reticulum (ER). This is an important, unresolved question in membrane biology. Discovering the molecular basis of flipping is essential to understanding how the phospholipid bilayer of biomembranes is propagated, and how the topologically complex syntheses of various glycolipids are executed. The latter processes are required for assembling biologically important cell surface molecules such as N-glycosylated and glycosylphosphatidylinositol (GPI)-anchored proteins in eukaryotes, and O-antigen-modified lipopolysaccharide in Gram-negative bacteria. (Glyco)phospholipid flip-flop does not occur at an appreciable rate in protein-free liposomes but occurs rapidly in the ER via a protein-dependent, metabolic energy-independent, bi-directional, facilitated diffusion process. We hypothesize that specific proteins, biogenic membrane flippases, facilitate the transbilayer diffusion of polar lipids in the ER, including the glycerophospholipids and isoprenoid-based glycolipids that are the focus of this proposal. The characteristics of lipid flip-flop in the ER rule out the participation of ABC transporters that have been identified as potential lipid translocators in other membrane settings. No biogenic membrane flippases have been identified that flip glycerophospholipids and isoprenoid-P-sugars, but compelling genetic evidence points to a membrane protein, Rft1p, as a flippase for dolichol-pyrophosphate based glycolipids in the ER-localized pathway of protein , N-glycosylation. We propose 2 specific aims: to characterize and identify a glycerophospholipid flippase from yeast and rat liver ER and to test biochemically the role of yeast Rft1p in flipping dolichol-PP-based glycolipids. These studies will make use of procedures that we have developed for the functional reconstitution and assay of lipid flip-flop in proteoliposomes generated from a detergent-extract of ER. We anticipate that our results will define a new class of membrane proteins for which no clear prototype currently exists and begin to address our eventual goal of obtaining a molecular definition of the mechanism of lipid flip-flop in biogenic membranes.

描述（由申请人提供）：本提案的长期目标是阐明极性脂质在生物（自我合成）膜（如内质网（ER））中翻转的机制。这是膜生物学中一个重要的、尚未解决的问题。发现翻转的分子基础对于理解生物膜的磷脂双层是如何繁殖的，以及各种糖脂的拓扑复杂合成是如何进行的至关重要。后一过程是组装生物学上重要的细胞表面分子所必需的，例如真核生物中的N-糖基化和糖基磷脂酰肌醇（GPI）锚定蛋白，以及革兰氏阴性细菌中的O-抗原修饰的脂多糖。（糖）磷脂翻转在无蛋白脂质体中不会以明显的速率发生，但在ER中通过蛋白依赖性、代谢能量独立性、双向、易化扩散过程快速发生。我们假设，特定的蛋白质，生物膜翻转酶，促进极性脂质在ER的跨双层扩散，包括甘油磷脂和类异戊二烯基糖脂，这是这个建议的重点。ER中脂质翻转的特征排除了ABC转运蛋白的参与，ABC转运蛋白已被确定为其他膜环境中的潜在脂质转运蛋白。没有生物膜翻转酶已被确定翻转甘油磷脂和类异戊二烯-P-糖，但令人信服的遗传证据指出，膜蛋白，Rft 1 p，作为一个翻转酶的长醇-焦磷酸基糖脂在ER定位的蛋白质，N-糖基化的途径。我们提出了2个具体的目标：从酵母和大鼠肝脏ER的甘油磷脂翻转酶的特征和确定和生化测试酵母RFT 1 P翻转长链-PP-为基础的糖脂的作用。这些研究将利用我们开发的程序，用于ER洗涤剂提取物产生的脂蛋白体中脂质翻转的功能重建和测定。我们预计，我们的研究结果将定义一类新的膜蛋白，目前还没有明确的原型存在，并开始，以解决我们的最终目标，获得一个分子定义的机制，脂质翻转生物膜。