Abstract
During in vitro homotypic yeast vacuole fusion Ca(2+) is transported into and out of the organelle lumen. In vitro, Ca(2+) is taken up from the medium by vacuoles upon the addition of ATP. During the docking stage of vacuole fusion Ca(2+) 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(2+) 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(2+) standards, up to 91 experimental conditions can be monitored in a single microplate using this method.