Phospholipid flip-flop in biogenic membranes

描述（申请人摘要）：在本R21（探索性/开发性）中 关于赠款申请，我们希望解决有关大会的一个中心问题 生物膜的磷脂双层，即，是什么 磷脂在生物体内的转运机制 （自我合成）膜？这是一个未被重视和研究的领域， 我们相信，尽管存在内在的实验困难， 测量磷脂触发器，我们处于一个独特的位置，以接近这一点， 长期存在的问题。新合成的磷脂位于 生物膜如内质网的细胞质小叶 (ER)真核细胞和细菌的细胞质膜（bCM）。而 这些脂质可以在它们所处的膜小叶中横向扩散 位于横向扩散或触发器的位置， 不会自发发生。然而，这种运动对于传播 膜双层，并且许多报告表明磷脂 快速穿过ER和BCM。这些报告还表明， 易位是双向的，通过易化扩散过程发生 不需要代谢能量输入。这一观察排除了 参与代谢的ABC家族转运蛋白 溶质和某些脂质的能量依赖性矢量转运。因此 磷脂跨ER转运的分子机制， bCM未知 我们假设存在一类新的脂质转运子， 以代谢能量独立的方式促进磷脂的扩散 穿过ER和bCM双层。我们称这些转运蛋白为生物膜 翻转酶，以区分它们与代谢能量需要的脂质 由ATP水解驱动的易位蛋白（如ABC转运蛋白）， 质子动力我们的目标是通过以下方法鉴定生物膜翻转酶： 生物化学和遗传学手段，从而提供了一个直接测试我们的假设。 为此，我们将探索生物化学重建/蛋白质纯化 使用芽孢杆菌膜作为起点的方法，以及向前和 利用大肠杆菌的反向遗传策略。其中最后一 涉及比较基因组学方法， 测序基因组数据库。 我们的长期目标是获得一个分子定义的机制， 磷脂的变化我们希望我们的分析不仅会影响 目前对膜生物发生的理解，也有助于 了解糖脂翻转事件，这是必不可少的组装 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.