Phospholipid flip-flop in biogenic membranes

描述(申请人摘要)：在本R21中(试探性/开发性) 批准申请，我们希望解决与大会有关的一个中心问题 生物膜的磷脂双层，即什么是 磷脂跨生物转运的机制 (自合成)膜？这是一个没有得到充分重视、研究不足的领域， 我们认为，尽管在实验中存在固有的困难 测量磷脂触发器，我们处于一个独特的位置来接近这一点 这是一个长期存在的问题。新合成的磷脂位于 内质网等生物膜的细胞质小叶 (ER)真核细胞和细菌的细胞膜(BCM)。而当 这些脂类可以在它们所在的膜片中横向扩散。 位于，横向扩散或触发器受热力学限制， 并不是自发发生的。然而，这样的运动对于传播是必不可少的 一种膜双层，许多报告表明磷脂 在ER和BCM之间快速转移。这些报告还表明， 移位是双向的，通过促进扩散过程发生 不需要新陈代谢能量输入。这一观察结果排除了 参与代谢的ABC转运蛋白家族的参与 依赖能量的，向量的溶质和某些脂类的运输。因此， 磷脂跨内质网和内质网转运的分子机制 BCM是未知的。 我们假设存在一类新的脂类转运体 促进磷脂以不依赖代谢能量的方式扩散 在ER和BCM双层上。我们称这些转位菌为生物原膜 翻转酶，以区别于代谢需要能量的脂类 转运体(如ABC转运体)由ATP水解物或 质子推动力。我们的目标是通过以下方法鉴定一种生物膜翻转酶 生化和遗传手段，从而为我们的假设提供了直接的检验。 为了做到这一点，我们将探索生化重组/蛋白质纯化 以芽孢杆菌膜为起点的方法，以及向前和 利用大肠杆菌的反向遗传策略。其中的最后一个 涉及比较基因组学方法，使可用的 测序的基因组数据库。 我们的长期目标是获得一个分子定义的机制 磷脂触发器。我们预计，我们的分析不仅会影响 目前对膜生物发生的理解，但也有助于 了解在组装过程中必不可少的糖脂触发事件 细胞表面成分。

DESCRIPTION (applicant's abstract): In this R21 (exploratory/developmental) grant application, we wish to address a central problem concerning the assembly of the phospholipid bilayer of biological membranes, i.e., what is the mechanism by which phospholipids are translocated across biogenic (self-synthesizing) membranes? This is an underappreciated, understudied area, and we believe that despite the intrinsic experimental difficulties in measuring phospholipid flip-flop, we are in an unique position to approach this longstanding problem. Newly synthesized phospholipids are located in the cytoplasmic leaflet of biogenic membranes such as the endoplasmic reticulum (ER) of eukaryotic cells and the cytoplasmic membrane (bCM) of bacteria. While these lipids can diffuse laterally in the membrane leaflet in which they are situated, transverse diffusion or flip-flop is thermodynamically restricted and does not occur spontaneously. However, such movement is essential to propagate a membrane bilayer, and a number of reports indicate that phospholipids translocate rapidly across the ER and bCM. These reports also indicate that translocation is bidirectional and occurs by a facilitated diffusion process requiring no metabolic energy input. This observation rules out the participation of the ABC family of transporters which are involved in metabolic energy-dependent, vectorial transport of solutes and some lipids. Thus the molecular mechanism by which phospholipids are translocated across the ER and bCM is unknown. We hypothesize that there exists a novel class of lipid translocators that facilitate diffusion of phospholipids in a metabolic energy-independent fashion across the ER and bCM bilayers. We term these translocators biogenic membrane flippases, to distinguish them from metabolic energy-requiring lipid translocators (like the ABC transporters) that are driven by ATP hydrolysis or protonmotive force. Our aim is to identify a biogenic membrane flippase by biochemical and genetic means, thus providing a direct test of our hypothesis. To do this we will explore biochemical reconstitution/protein purification approaches using Bacillus membranes as a starting point, as well as forward and reverse genetic strategies utilizing Escherichia coli. The last of these involves a comparative genomics approach, making novel use of available sequenced genome databases. Our long term goal is to obtain a molecular definition of the mechanism of phospholipid flip-flop. We expect that our analyses will not only impact current understanding of membrane biogenesis, but also contribute to an understanding of glycolipid flip-flop events that are essential in the assembly of cell surface components.