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Dissecting the mechanism of Ca 2+ ‐triggered membrane fusion: Probing protein function using thiol reactivity
Author(s) -
Furber Kendra L,
Dean Kwin T,
Coorssen Jens R
Publication year - 2010
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.2009.05278.x
Subject(s) - lipid bilayer fusion , fusion protein , chemistry , thiol , membrane curvature , fusion , biochemistry , membrane protein , biophysics , function (biology) , membrane , microbiology and biotechnology , vesicle , biology , recombinant dna , linguistics , philosophy , gene
Summary 1. Ca 2+ ‐triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage‐specific native preparation fully enabling the directly coupled functional–molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol‐ and sphingomyelin‐enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca 2+ transient. 3. There is less known about the identity of proteins involved in the Ca 2+ ‐triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol‐reactive reagents have identified different effects on Ca 2+ sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca 2+ sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca 2+ sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol‐enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins.