Analysis of the molecular function of sterols in membrane fusion

膜融合对于细胞器功能、激素分泌、细胞生长和神经传递是必不可少的。从酵母到人类的膜融合需要相同的保守蛋白质和脂质，包括固醇。虽然介导融合的蛋白质已被广泛研究，但对脂质的功能知之甚少。空泡（溶酶体的酵母等效物）的融合已经通过分离的空泡之间的融合的体内遗传学和体外测定进行了广泛的研究。甾醇对于液泡在体内和与分离的细胞器的融合是必需的，并且对于过氧化物酶体、质膜和胞吐运输囊泡的融合也是必需的。尽管如此，每个细胞器的化学复杂性，甾醇在每个细胞膜中的普遍存在和甾醇的多种功能，迄今为止，排除了甾醇如何支持融合的研究。Wickner教授的实验室最近实现了蛋白脂质体膜融合与所有纯化和化学定义的液泡成分的重建，由13种重组蛋白和8种脂质组成。这种重组融合需要甾醇。两个因素使得这是一个无与伦比的机会来解开甾醇在融合中的作用：1。在该系统中随意控制固醇水平的能力，无论是在蛋白脂质体制备过程中还是通过随后用合成固醇载体甲基-β-环糊精进行固醇“缓冲”，以及2.在宿主实验室中常规进行的膜融合每个步骤的快速定量测定。为了确定需要甾醇的步骤，将在严格控制甾醇水平的情况下进行这些阶段特异性试验。了解甾醇的作用对所有的融合反应都很重要。学习这个实验室的还原论方法对我未来计划的线粒体膜融合的生化分析很重要。

Membrane fusion is essential for organelle function, hormone secretion, cell growth, and neurotransmission. The same conserved proteins and lipids, including sterol, are required for membrane fusion from yeast to humans. While the proteins which mediate fusion have been studied extensively, little is known about the function of lipids. The fusion of vacuoles, the yeast equivalent of lysosomes, has been extensively studied through in vivo genetics and in vitro assays of the fusion between isolated vacuoles. Sterol is essential for the fusion of vacuoles, in vivo and with the isolated organelle, and is also required for peroxisome, plasma membrane, and exocytic trafficking vesicle fusion. Nonetheless, the chemical complexity of each organelle, the ubiquitous presence of sterols in each cell membrane and the multiple functions of sterols have heretofore precluded studies of how sterols support fusion. Professor Wickner's lab has recently achieved a reconstitution of proteoliposomal membrane fusion with all purified and chemically defined vacuolar components, consisting of 13 recombinant proteins and 8 lipids. Sterol is required for this reconstituted fusion. Two factors make this an unparalleled opportunity to unravel the role of sterols in fusion: 1. The capacity in this system to control the levels of sterol at will, either during proteoliposome preparation or by subsequent sterol "buffering" with the synthetic sterol carrier methyl-beta-cyclodextrin, and 2. The rapid and quantitative assays of each step of membrane fusion which are routine in the host lab. To establish the steps where sterol is needed, these stage-specific assays will be performed with rigorously controlled levels of sterol. Understanding the roles of sterols will be important for all fusion reactions. Learning this lab's reductionist approach will be important for me for my future planned biochemical analysis of the fusion of mitochondrial membranes.