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

1. Ca2+-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. Additionally, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca2+ transient. 3. There is less known concerning the identity of proteins involved in the Ca2+-triggering steps of membrane fusion. Thiol-reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents identify different effects on Ca2+-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 Ca2+ sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca2+-sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was employed 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 labeled proteome, narrowing the number of candidate proteins