Analysis of the molecular function of sterols in membrane fusion

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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.