Using Thiol-Reactivity to Identify Proteins Involved in the Ca2+-Triggering Steps of Native Membrane Fusion

Abstract

Ca2+-triggered membrane fusion, the defining step of exocytosis, enables temporal/spatial control over the release of biologically active compounds. The mechanism by which Ca2+ triggers and modulates native membrane fusion is still poorly understood. As an unbiased approach to investigating this process, the effects of several thiol-reactive reagents on the homotypic fusion of isolated cortical vesicles (a stage-specific preparation for analyses of native Ca2+-triggered fusion) have been characterized. Such reagents have been consistently shown to inhibit the Ca2+-sensitivity, rate and extent of triggered fusion. However, we recently showed that iodoacetamide can also potentiate the Ca2+-sensitivity and rate of release [1]. This implicates two distinct thiol sites in the fusion process - one involved in the ability of vesicles to fuse (extent) and one that modulates fusion efficiency (Ca2+-sensitivity and kinetics). Capitalizing on this potentiating effect, we have now identified other fluorescent thiol-reactive reagents with similar effects: treatment with Lucifer yellow iodoacetamide, monobromobimane or dibromobimane resulted in an average leftward shift in EC50 from 17.2±1.6μM to 8.9±1.9μM [Ca2+]free. These fluorescent reagents can be used to enhance fusion and label proteins involved in the Ca2+-sensing mechanism. The lipid matrix at or near the fusion site can also modulate the fusion process, specifically via cholesterol- and sphingomyelin-enrichment that is thought to regulate the Ca2+-sensitivity and rate of fusion through spatial organization of critical lipids and proteins [2,3]. Proteins involved in Ca2+-sensing are thus likely to be situated within such areas of the membrane. Isolation of fluorescently labeled proteins from cholesterol-enriched vesicle membrane fractions by 2-dimesional electrophoresis is now being used to identify proteins potentially involved in the Ca2+-triggering steps of membrane fusion.1) J. Chem. Biol., Epub 3/10/08. 2) J. Cell Sci. 2005, 118:4833. 3) J. Cell Sci. 2006, 119:2688.