GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis
GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis作者机构：Instituto de Histologia y Embriologia IHEM-CONICET Facultad de Ciencias Medicas Universidad Nacional de Cuyo Mendoza Argentina Department of Biophysics UT Southwestern Medical Center Dallas TX USA Servicio de Patologla Hospital Italiano de Buenos Aires Buenos Aires Argentina
出 版 物：《Journal of Molecular Cell Biology》 (分子细胞生物学报（英文版）)
年 卷 期：2014年第8卷第4期
页 面：286-298页
核心收录：
学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 09[农学] 0901[农学-作物学]
基 金：ConsejoNacionaldeInvestigacionesCientificasyTecnicas(grantnumberPIP2038toL.S.M.) AgenciaNacionaldePromocionCientificayTecnologica(grantnumbersPICT2006-1036,PICT2010-0342toC.N.T.)
主 题：Rab3 cycle GTP hydrolysis exocytosis acrosome calcium sperm
摘 要：Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizingthe exocytotic response of streptolysin O-permeabiUzed human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranyigeranyiated, and active Rab3A elicited human sperm exocytosis perse. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its *** prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
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