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SNAREs pp 253-262 | Cite as

Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles

  • Massimo D’Agostino
  • Andreas Mayer
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

Fluorescence de-quenching can be used to analyze membrane lipid mixing during an in vitro fusion reaction. Here we describe a method to measure lipid mixing using vacuolar membranes purified from the yeast Saccharomyces cerevisiae. Labeling the isolated organelles with rhodamine-phosphatidylethanolamine allows to reveal ATP-dependent lipid mixing through fluorescence de-quenching in a spectrofluorometer. Combining this assay with content mixing indicators, such as the fusion-dependent maturation of a luminal vacuolar phosphatase, then permits the detection of hemifusion intermediates and the analysis of the requirements for fusion pore opening.

Key words

Membrane fusion Lipid mixing Yeast Vacuole 

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Copyright information

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

Authors and Affiliations

  1. 1.Département de BiochimieUniversité de LausanneEpalingesSwitzerland

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