Regulated secretion: SNARE density, vesicle fusion and calcium dependence

Abstract

SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion(Ca2+ sensitivity and the catalysis and execution of fusion)require additional proteins that function downstream of SNAREs.

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

“…CSC and CV preparations CV-PM (cell surface complex; CSC) and CV preparations were isolated from unfertilized sea urchin (Srongylocentrotus purpuratus, Westwind, BC, Canada) eggs as previously described [6][7][8][9][14][15][16]. All treatments and fusion assays were carried out in baseline intracellular medium (BIM; 210 mM potassium glutamate, 500 mM glycine, 10 mM NaCl, 10 mM Pipes, 0.05 mM CaCl 2 , 1 mM MgCl 2 , 1 mM EGTA; pH 6.7; [23]) supplemented with 2.5 mM ATP and protease inhibitors, unless otherwise stated.…”

“…The system can be further refined as isolated CV undergo fast, Ca 2+ -triggered fusion with one another [6][7][8][9][10][11][12][13][14][15][16] and even with pure lipid membranes [12,17], indicating that CV contain the minimal essential molecular components for intermembrane attachment, Ca 2+ sensing, and membrane fusion. CV are thus a high purity, robust, stage-specific preparation of release-ready secretory vesicles with which to analyze the mechanisms essential to Ca 2+ -triggered membrane fusion [6,7,[14][15][16].…”

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

“…CSC and CV preparations, treatments, and fusion assays CSC/CV were isolated from fresh and 20-h incubated eggs using standard protocols [27,30,31]. Acute treatments and fusion assays were carried out in baseline intracellular media (BIM-210 mM potassium glutamate, 500 mM glycine, 10 mM NaCl, 10 mM 1,4-piperazinediethanesulfonic acid (PIPES), 1 mM MgCl 2 , 0.05 mM CaCl 2 , 1 mM ethyleneglycoltetraacetic acid (EGTA); pH 6.7) with the addition of protease inhibitors and 2.5 mM adenosine triphosphate (ATP) [30].…”

“…Acute treatments and fusion assays were carried out in baseline intracellular media (BIM-210 mM potassium glutamate, 500 mM glycine, 10 mM NaCl, 10 mM 1,4-piperazinediethanesulfonic acid (PIPES), 1 mM MgCl 2 , 0.05 mM CaCl 2 , 1 mM ethyleneglycoltetraacetic acid (EGTA); pH 6.7) with the addition of protease inhibitors and 2.5 mM adenosine triphosphate (ATP) [30]. Cinnamycin was delivered to CV suspensions (optical density (OD) 405 0.4) from DMSO/BIM (1:9) stocks, while neomycin was delivered from an aqueous solution (OD 405 0.4).…”

“…Shearing yields cell surface complexes (CSC)-plasma membrane (PM) sheets with docked, fusion-ready cortical vesicles (CV), from which high purity CV are isolated [29,31,85,96]. Unlike mammalian secretory vesicles, CV retain Ca 2+ sensitivity and fusion competence in vitro hours after isolationwashed free of soluble components, CV are 'locked' in the docked, fusion-ready state, requiring only an increase in [Ca 2+ ] free to trigger fusion [30,57,75,80,90]. Being amenable to biochemical manipulations, CSC and CV have proven invaluable in dissecting molecular mechanisms underlying docking, Ca 2+ -triggering, and membrane fusion; comparable quantitative manipulations are unfeasible in other secretory systems [30,76,97].…”