Real-Time Fluorescence Detection of Calcium Efflux During Vacuolar Membrane Fusion.

During in vitro homotypic yeast vacuole fusion Ca is transported into and out of the organelle lumen. In vitro, Ca is taken up from the medium by vacuoles upon the addition of ATP. During the docking stage of vacuole fusion Ca is effluxed from the lumen upon the formation of trans-SNARE complexes between vesicles. Here we describe a real-time fluorescence-based assay to monitor the transport of this cation using purified organelles. Extraluminal Ca is detected when the cation binds the low-affinity fluorescent dye Fluo-4 dextran. This allows for the use of a 96-well microtiter plate to be read in a fluorescence plate reader. Thus, in addition to a curve of calibrated Ca standards, up to 91 experimental conditions can be monitored in a single microplate using this method.