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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Clapham DE (2007) Calcium signaling. Cell 131:1047–1058
Chapman ER (2008) How does synaptotagmin trigger neurotransmitter release. Annu Rev Biochem 77:615–641
Peters C, Mayer A (1998) Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion. Nature 396:575–580
Dunn T, Gable K, Beeler T (1994) Regulation of cellular Ca2+ by yeast vacuoles. J Biol Chem 269:7273–7278
Merz AJ, Wickner W (2004) Trans-SNARE interactions elicit Ca2+ efflux from the yeast vacuole lumen. J Cell Biol 164:195–206
Starai VJ, Thorngren N, Fratti RA, Wickner W (2005) Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae. J Biol Chem 280:16754–16762
Takita Y, Engstrom L, Ungermann C, Cunningham KW (2001) Inhibition of the Ca(2+)-ATPase Pmc1p by the v-SNARE protein Nyv1p. J Biol Chem 276:6200–6206
Sasser TL, Padolina M, Fratti RA (2012) The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation. Biochem J 448:365–372
Sasser TL, Lawrence G, Karunakaran S, Brown C, Fratti RA (2013) The yeast ABC transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion. J Biol Chem 288:18300–18310
Miner GE, Starr ML, Hurst LR, Sparks RP, Padolina M, Fratti RA (2016) The central polybasic region of the soluble SNARE (soluble N-Ethylmaleimide-sensitive factor attachment protein receptor) Vam7 affects binding to phosphatidylinositol 3-phosphate by the PX (Phox homology) domain. J Biol Chem 291:17651–17663
Miner GE, Starr ML, Hurst LR, Fratti RA (2017) Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity. Traffic 18:315–329
Scott JH, Schekman R (1980) Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol 142:414–423
Slusarewicz P, Xu Z, Seefeld K, Haas A, Wickner WT (1997) I2B is a small cytosolic protein that participates in vacuole fusion. Proc Natl Acad Sci U S A 94:5582–5587
Acknowledgments
This work was supported in part by NIH grant GM101132 to RAF.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8760-3_21
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8759-7
Online ISBN: 978-1-4939-8760-3
eBook Packages: Springer Protocols