Phospholipid Flip-flop in Biogenic Membranes

描述（由申请人提供）：脂质从生物膜的一侧到另一侧的双向移位被称为触发器。脂质翻转通过内质网（ER）膜是蛋白质n -糖基化和gpi锚定所必需的。这些蛋白质修饰在真核生物中是必不可少的；例如，它们的基因废除会导致哺乳动物的胚胎死亡，并使酵母菌无法存活。跨内质网的脂质翻转也是膜生物发生所必需的：在内质网细胞质面合成的磷脂必须转移到相反的面，以使膜双分子层均匀生长。当内质网膜扩张和糖蛋白分泌增加时，内质网脂质翻转的需求可能异常高；例如，在B淋巴细胞向分泌抗体的浆B细胞分化的过程中就会发生这种情况。由于带极性脂质头基团通过膜疏水内部的能量障碍，无辅助的翻转非常缓慢，然而脂质在几秒钟的时间尺度上迅速地翻转穿过内质网膜。这是因为内质网拥有特定的转运蛋白（翻转酶），可以加速脂质翻转到生理上足够的速度。内质网中的脂质翻转是通过一种不依赖于atp的机制发生的，在这种机制中，翻转促进了脂质的“下坡”运输；这将内质网翻转酶与其他易位子区别开来，这些易位子通常在真核质膜中发现，它们将ATP水解与脂质的集中“上坡”运输结合起来。我们估计有多达六种不同的内质网脂质翻转酶，但没有一种在分子水平上被鉴定出来。我们开发了生化重组系统，概括了内质网膜双层扩张和蛋白质糖基化所需的三种翻转酶的活性。这些翻转酶特异性地转运甘油磷脂、低聚糖二磷酸多醇和甘露糖磷酸多醇。我们的目标是识别这些生理上重要的易位子，并了解其作用机制的长期目标。我们建议通过双管齐下的方法来做到这一点，一方面涉及蛋白质纯化和质谱分析，另一方面涉及酵母ER膜蛋白的系统收集筛选。我们的纯化工作将有助于使用新的亲和矩阵。我们还将使用部分纯化的翻转酶制剂来继续我们的努力，以确定这些蛋白质的特异性。我们发表的工作和初步数据使我们处于实现这些目标的绝佳位置。

DESCRIPTION (provided by applicant): The bi-directional translocation of lipids from one side of a biological membrane to the other is termed flip-flop. Lipid flip-flop across the endoplasmic reticulum (ER) membrane is required for protein N-glycosylation and GPI-anchoring. These protein modifications are essential in eukaryotes; for example, their genetic abrogation causes embryonic lethality in mammals and renders yeast unviable. Lipid flip-flop across the ER is also required for membrane biogenesis: phospholipids that are synthesized on the cytoplasmic face of the ER must be translocated to the opposite face to enable the membrane bilayer to grow uniformly. The demand for lipid flip-flop at the ER is likely to be exceptionally high when the ER membrane expands and glycoprotein secretion increases; this occurs, for example, during the differentiation of B-lymphocytes to antibody-secreting plasma B cells. Unassisted flip-flop is extremely slow because of the energy barrier to taking the polar lipid head group through the hydrophobic interior of the membrane, yet lipids flip-flop rapidly across the ER membrane on a time-scale of seconds. This is because the ER possesses specific transport proteins (flippases) that accelerate lipid flipping to a physiologically sufficient rate. Lipid flipping in the ER occurs by an ATP-independent mechanism in which the flippases facilitate 'downhill' transport of lipids; this distinguishes ER flippases from other translocators, typically found in the eukaryotic plasma membrane, that couple ATP hydrolysis to concentrative 'uphill' transport of lipids. We estimate that there are as many as six different ER lipid flippases but none of these have been identified at the molecular level. We developed biochemical reconstitution systems that recapitulate the activity of three of the flippases required for ER membrane bilayer expansion and protein glycosylation. These flippases specifically translocate glycerophospholipids, oligosaccharide diphosphate dolichols and mannose-phosphate dolichol. Our aim is to identify these physiologically important translocators with the long-term goal of understanding their mechanism of action. We propose to do this via a two-pronged approach involving protein purification and mass spectrometry on the one hand, and screening of systematic collections of yeast ER membrane proteins on the other. Our purification efforts will be aided by the use of novel affinity matrices. We will also use partially purified flippase preparations to continue our efforts to define the specificity of these proteins. Our published work and preliminary data put us in an excellent position to accomplish these aims.