TY - JOUR
T1 - Ca2+-induced recruitment of the secretory vesicle protein DOC2B to the target membrane
AU - Groffen, Alexander J.A.
AU - Brian, Elisabeth C.
AU - Dudok, Jeroen J.
AU - Kampmeijer, Joris
AU - Toonen, Ruud F.
AU - Verhage, Matthijs
PY - 2004/5/28
Y1 - 2004/5/28
N2 - Ca2+-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ 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 Ca2+ 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 Ca2+-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 Ca2+ 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 Ca2+-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.
AB - Ca2+-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ 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 Ca2+ 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 Ca2+-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 Ca2+ 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 Ca2+-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.
UR - http://www.scopus.com/inward/record.url?scp=2542453015&partnerID=8YFLogxK
U2 - https://doi.org/10.1074/jbc.M400731200
DO - https://doi.org/10.1074/jbc.M400731200
M3 - Article
C2 - 15033971
SN - 0021-9258
VL - 279
SP - 23740
EP - 23747
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 22
ER -