Phospholipid 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（自合成）膜（例如内质网（ER））上翻转的机制。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如何传播以及如何执行各种糖脂的拓扑复杂合成至关重要。后一个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）磷脂翻转在无蛋白质脂质体中不会以明显的速率发生，但在 ER 中通过蛋白质依赖性、代谢能量独立、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）促进内质网中极性脂质的跨双层扩散，包括本提案重点关注的甘油磷脂和类异戊二烯糖脂。内质网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潜在的脂质易位蛋白。目前还没有发现生物膜翻转酶能够翻转甘油磷脂和类异戊二烯-P-糖，但令人信服的遗传证据表明，膜蛋白 Rft1p 是内质网蛋白 N-糖基化途径中基于多甘醇焦磷酸的糖脂的翻转酶。我们提出了 2 个具体目标：表征和鉴定来自酵母和大鼠肝脏 ER 的甘油磷脂翻转酶，并通过生化方法测试酵母 Rft1p 在翻转基于 dolichol-PP 的糖脂中的作用。这些研究将利用我们开发的程序，用于对由 ER 洗涤剂提取物产生的蛋白脂质体中的脂质触发器进行功能重建和测定。我们预计我们的结果将定义一类目前尚不存在明确原型的新膜蛋白，并开始解决我们的最终目标，即获得生物膜中脂质翻转机制的分子定义。

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.