Abstract
During in vitro homotypic yeast vacuole fusion Ca2+ is transported into and out of the organelle lumen. In vitro, Ca2+ is taken up from the medium by vacuoles upon the addition of ATP. During the docking stage of vacuole fusion Ca2+ 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 Ca2+ 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 Ca2+ standards, up to 91 experimental conditions can be monitored in a single microplate using this method.
Key words
- Membrane fusion
- SNARE
- Membrane trafficking
- Ca2+ efflux
- Fluorescence
- Yeast vacuole
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Acknowledgments
This work was supported in part by NIH grant GM101132 to RAF.
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Miner, G.E., Fratti, R. (2019). Real-Time Fluorescence Detection of Calcium Efflux During Vacuolar Membrane Fusion. In: Fratti, R. (eds) SNAREs. Methods in Molecular Biology, vol 1860. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8760-3_21
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DOI: https://doi.org/10.1007/978-1-4939-8760-3_21
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