Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 32, Pages 13010-13015Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0700970104
Keywords
GTP gamma S; hemifusion; membrane fusion; SNARE; yeast vacuole
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Membrane fusion entails organelle docking and subsequent mixing of membrane bilayers and luminal compartments. We now present an in vitro assay of fusion, using yeast vacuoles bearing domains of either Fos or Jun fused to complementary halves of)beta-lactamase. Upon fusion, these proteins associate to yield beta-lactamase activity. This assay complements the standard fusion assay (activation of pro-Pho8p in protease-deficient vacuoles by proteases from pho8 Delta vacuoles). Both the beta-lactamase and proPho8p activation assays of fusion show the same long kinetic delay between SNARE pairing and luminal compartment mixing. Lipid-mixing occurs rapidly after SNARE pairing but well before aqueous compartment mixing. These results support a model in which SNARE pairing leads to rapid hemifusion, followed by slow further lipid rearrangement and aqueous compartment mixing.
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