Study of the role of Rab3A in the secretory pathway

• Main Authors: Chien-Chang Huang, 黃建彰
• Other Authors: Lung-Sen Kao
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/n5k8cj

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 98 === Rab3A is a small G protein of Rab family that involves in the late steps of exocytosis, including docking, priming and fusion. However, it is not clear at which step of exocytic pathway that Rab3A actually acts. Near the plasma membrane, Rab3A/RIM/Munc13-1 form a tripartite complex which is thought to regulate the priming of secretory vesicles. Phorbol ester, PMA, is known to increase the number of primed vesicles. It activates PKC-independent Munc13-1 activation and PKC- dependent Munc18-1-mediated interaction with syntaxin 1a to enhance vesicle priming. In this study, we examined the effects of PMA on ATP-stimulated exocytosis in PC12 cells to study the possible molecular interactions among Rab3A, Munc13-1 and Munc18-1. PMA enhanced the ATP-induced vesicular release and accelerated the kinetics of vesicle release. Our results show the effects of PMA varied in cells overexpressing Rab3A and its mutants. In Rab3A-knockdown cells, overexpression of Munc13-1 inhibited the secretion completely and the inhibition was rescued by RIM-binding deficient Munc13-1 mutant, 128-Munc13-1. In 128-Munc13-1 overexpressing cells, the effects of Rab3A on secretion was abolished and the effect of PMA disappeared in cells overexpressing GTP-Rab3A (Q81L) which could be reversed by co-expressing Munc18-1. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Munc18-1 was shown to interact with Rab3A and promoted the disassociation of Rab3A upon ATP stimulation. In summary, the Rab3A cycle is coupled with the activation of Munc13-1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18-1 acts downstream of Munc13-1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18-1 promotes Rab3A dissociation from the vesicles, which then results in fusion.