Ca2+-induced Recruitment of the Secretory Vesicle Protein DOC2B to the Target Membrane

Abstract

Ca 2؉ -dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca 2؉ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca 2؉ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca 2؉ -dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca 2؉ -induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca 2؉ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca 2؉ -induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.Ca 2ϩ -induced exocytosis is a widely conserved mechanism of importance for a broad range of secretory systems, such as the release of neurotransmitters and neuropeptides in the central and peripheral nervous system. To attain fusion competence, secretory vesicles must first dock at the target membrane and undergo a process called priming. The release of fusion-ready vesicles is coupled to the opening of Ca 2ϩ channels in the plasma membrane, producing a transient high Ca 2ϩ concentration (10 -100 M for ϳ1 ms) in close proximity to the channels. The inward Ca 2ϩ current then diffuses into the cytoplasm, resulting in a residual concentration in the range of 1 M during tens of seconds. It is widely accepted that this residual Ca 2ϩ concentration induces short term changes in exocytotic strength (e.g. see Refs. 1 and 2), although the mechanisms that contribute to this phenomenon remain largely unclear. As a result, the secretory strength of neuronal and endocrine release sites is modulated in an activity-dependent way. This phenomenon is important to avoid vesicle depletion during repetitive stimulation and is furthermore considered in neurons to contribute to memory and learning.Although Ca 2ϩ is the most important messenger after high frequency activation of the release site, exocytotic potentiation can also be induced by diacylglycerol (synthesized by members of the phospholipase family) or by its phorbolester analogues. Diacylglycerol has multiple intracellular targets (e.g. protein kinase C isoforms), but the potentiating effect in regulated exocytosis is mediated by the priming factor Munc13-1. Munc13-1-deficient mice die shortly after birth and show a defect in synapt...

Doc2β Is a Novel Munc18c-interacting Partner and Positive Effector of Syntaxin 4-mediated Exocytosis

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The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets

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