Abstract

All cells and organelles must regulate volume to control their size, shape and copy number. Volume homeostasis, in turn, is dependent on osmosis facilitated by aquaporins. Despite being critical for membrane fusion and fission events that underlie organelle morphology and membrane trafficking, volume regulations in the endocytic pathway and the aquaporins responsible have not been characterized. Thus, the primary goals of my dissertation research were to better understand the role of osmosis in membrane fusion or fission and identify the aquaporin(s) responsible using the yeast vacuole as a model. First, I characterized osmosis across isolated vacuole membranes and identified the aquaporins responsible, Aqy3 and Fps1, using stopped-flow fluorometry. Fluorescence microscopy revealed that knocking out AQY3 or both aquaporins disrupted vacuole morphology, indicative of an impairment in membrane fission. Using a new cell-free vacuole fission assay, I then determined that osmosis can drive fission in vitro and that this process involves inactivation of Ytp7, a Rab-GTPase and key regulator of vacuole morphology. These results support a model whereby water efflux by Aqy3 permits membrane invagination while signaling the fission machinery necessary for membrane fission.