Dissecting the mechanism of Ca2+-triggered membrane fusion: probing protein function using thiol reactivity

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.