Abstract
Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.
Highlights
Regulated exocytosis is a fundamental process for the release of bioactive molecules from cells.Several discrete stages are involved in the exocytotic pathway, including trafficking of secretory vesicles containing cellular cargo to the appropriate target membrane, tethering and docking for attachment of secretory vesicles to release sites, priming of the vesicles to render them fusion competent, Ca2+sensing and triggering events to regulate and facilitate fusion of the secretory vesicle and plasma membranes (PM), and merger of the two lipid bilayers to form a fusion pore releasing vesicular contents
We have previously shown the feasibility of this approach with the fluorescent thiol-reagent, Lucifer yellow iodoacetamide (LYIA) [40]
The thiol reagents used have a biphasic effect, with low concentrations potentiating fusion parameters and high concentrations inhibiting all parameters of Ca2+ -triggered membrane fusion [39,40]
Summary
Sensing and triggering events to regulate and facilitate fusion of the secretory vesicle and plasma membranes (PM), and merger of the two lipid bilayers to form a fusion pore releasing vesicular contents. This involves the coordinated actions of both lipid and protein components of the secretory vesicle membrane, the later steps of Ca2+ -triggered exocytosis. There remains debate regarding the identity of proteins involved in the Ca2+ -sensing and triggering steps that regulate the efficiency of exocytosis.
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