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

BUSTOS, MATÍAS A.; ROGGERO, CARLOS M.; DE LA IGLESIA, PAOLA X.; MAYORGA, LUIS S.; TOMES, CLAUDIA N.

Journal of Molecular Cell Biology

Oxford University Press

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

1674-2788

Exocytosis of mammalian sperm dense-core secretory granule relies on thesame fusion molecules as all other secretory cells; one suchmolecule is the small GTPase Rab3A. Here,wereport an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizingthe exocytotic response of streptolysin O-permeabilizedhumanspermto the acute application of anumberofRab3A-containingconstructs and correlating the findings with those gathered with the endogenous protein. Full length, geranylgeranylated, and activeRab3A elicited human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminaldomain of theRab3Amoleculewas necessaryandsufficient to promoteexocytosis, whereas its amino-terminus prevented calcium-triggeredsecretion. 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 acrosomalstaining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, andfound that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishingthat Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.