title: Involvement of Rab3A in vesicle priming during exocytosis: interaction with Munc13-1 and Munc18-1.
abstract: Rab3A is a small G-protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13-1 and Munc18-1 during vesicle priming. Phorbol 12-myristate 13-acetate (PMA) is known to enhance the percentage of fusion-competent vesicles and this is mediated by protein kinase C (PKC)-independent Munc13-1 activation and PKC-dependent dissociation of Munc18-1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13-1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab-interacting molecule (RIM)-binding deficient Munc13-1 mutant, 128-Munc13-1, the effects of Rab3A on PMA-induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP-Rab3A (Q81L), could be reversed by co-expressing Munc18-1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18-1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that 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 vesicles, which then results in fusion.
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, R. O. C.
authors.forename: Chien-Chang
authors.initials: CC
authors.lastname: Huang
authors.forename: De-Ming
authors.initials: DM
authors.lastname: Yang
authors.forename: Chung-Chih
authors.initials: CC
authors.lastname: Lin
authors.forename: Lung-Sen
authors.initials: LS
authors.lastname: Kao
mesh_terms: D000818:Animals; D005089:Exocytosis / Q000502:physiology*; D018613:Microscopy, Confocal; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D038761:Photobleaching; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection; D020826:rab3A GTP-Binding Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C497939:Stxbp1 protein, rat; C103912:Unc13a protein, rat; D020826:rab3A GTP-Binding Protein