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）：该提案的长期目标是阐明极性脂质在生物源性（自合成的）膜（例如内质网）（ER）等生物源性（自合成）膜中的机制。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如何传播以及如何执行各种糖脂的拓扑复杂合成。后一个过程是在真核生物中组装生物学上重要的细胞表面分子（例如N-糖基化和糖基磷脂酰肌醇（GPI）锚定的蛋白质，以及O-抗原修饰的脂多糖在革兰氏阴性细菌中的蛋白质。 （Glyco）在无蛋白质脂质体中并不以明显的速度发生磷脂触发器，而是通过蛋白质依赖性的，代谢独立的，双向的，促进的扩散过程在ER中迅速发生。我们假设特定的蛋白质，生物膜氟酶促进了极性脂质在ER中的跨贝元扩散，包括甘油磷脂和基于类异胞二糖的甘油类糖脂，这是该建议的重点。在ER中，脂质触发器的特征排除了ABC转运蛋白的参与，这些ABC转运蛋白已被确定为其他膜环境中潜在的脂质转运剂。尚未鉴定出尚未鉴定出fl甘油磷脂和异磷酸 - 糖 - 糖，但令人信服的遗传证据表明膜蛋白RFT1P是Dolichol-pyprophophatre-磷酸磷酸磷酸糖磷脂的flippase，在ER蛋白质中，N-糖蛋白，N-糖蛋白，N-糖蛋白质，N-糖蛋白质，N-糖蛋白，n-糖蛋白，N-糖蛋白，N-糖蛋白质，n-糖蛋白，N-糖蛋白质，n-糖蛋白，n-糖蛋白含量。我们提出了2个特定目的：表征和鉴定酵母和大鼠肝ER的甘油磷脂flippase，并以生化测试酵母RFT1p在拨动基于Dolichol-PP的糖脂中的作用。这些研究将利用我们为通过ER的清洁剂提取的蛋白质脂质体中的脂质触发器的功能重构和测定而开发的程序。我们预计我们的结果将定义一类新的膜蛋白，目前不存在清晰的原型，并开始解决我们最终的目标，即在生物膜中获得脂质触发器机制的分子定义。