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

Año: 2014 vol. 6 p. 286 - 298

1674-2788

Exocytosis of mammalian sperm´s acrosome ? a very large dense-core secretory granule ? relies on the same fusion molecules as neuronal, endocrine, and all other secretory cells. One such molecule is the small GTPase Rab3A. It has been known for some time that recombinant, full length Rab3A, geranylgeranylated and loaded with GTP, elicits human sperm exocytosis per se. Here, we report a deeper 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 Rab3Acontaining constructs. We found that Rab3A exerts either a positive or a negative functions on acrosomal release depending on the exocytotic stage reached by the system at the time of addition. Thus, Rab3A promotes exocytosis when introduced into sperm early but prevents it if added after the endogenous machinery has achieved docking of the acrosome to the plasma membrane. By means of Rab3A/Rab22A chimeric proteins, we demonstrate that these seemingly opposite regulatory properties are segregated to different domains of the Rab3A molecule, with the carboxy-terminus being responsible for promoting, and the amino-terminus for inhibiting, exocytosis. Unlike triggering, which requires geranylgeranylation and activation by GTP or a non-hydrolyzable GTP analogue, inhibition does not need isoprenylation but depends on Rab3A?s inability to hydrolyze GTP. These findings led us to scrutinize in more detail the behaviour of endogenous protein during the exocytotic cascade. Rab3 first exchanges GDP for GTP and later hydrolyzes bound GTP during the exocytotic cascade; all these changes take place in the acrosomal region of the cell. This sequential activation and inactivation correlates with the sensitivity of the system to Rab-GDI and is mandatory to accomplish exocytosis. Our findings contribute to clarify the many controversies about the role of Rab3A in secretion by establishing that this small G protein modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage at which it is studied.