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doi: 10.1242/10.1242/jcs.00374

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Regulated secretion: SNARE density, vesicle fusion and calcium dependence

¹ Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA
² Unit on Biologic Computation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA
³ Department of Physiology and Biophysics, Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

^* Authors for correspondence (e-mail: jcoorsse{at}ucalgary.ca or joshz{at}helix.nih.gov)

Accepted 20 January 2003

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.

Key words: Secretory vesicles, Exocytosis, Membrane fusion, Sea urchins, Quantitative immunoblotting

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