GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis

BUSTOS MA; ROGGERO CM; DE LA IGLESIA P; MAYORGA LS; TOMES CN

Journal of Molecular Cell Biology

Oxford University Press

Lugar: Oxford; Año: 2014 vol. 6 p. 286 - 298

1674-2788

Exocytosis of mammalian sperm´s 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 a deep biochemical characterization of Rab3A?s role in secretion by scrutinizing the exocytotic response of streptolysin O-permeabilized 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, geranylgeranylated and active Rab3A elicits human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins we demonstrate that the carboxy-terminus domain of the Rab3A molecule is necessary and sufficient to promote exocytosis whereas its amino-terminus prevents 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 Rab3A?s inability 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 establish that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.