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.
Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles / D'Agostino, Massimo; Mayer, Andreas. - 1860:(2019), pp. 253-262. [10.1007/978-1-4939-8760-3_16]
Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles
D'Agostino, Massimo;
2019
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
