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SNAREs pp 323-331 | Cite as

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

  • Gregory E. Miner
  • Rutilio Fratti
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

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 

Notes

Acknowledgments

This work was supported in part by NIH grant GM101132 to RAF.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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