PERSISTENTLY ACTIVE RAB3A, AND A CHIMAERIC PROTEIN CONTAINING ITS AMINO-TERMINAL PORTION, PREVENT THE LATE STAGES OF SPERM EXOCYTOSIS BY STABILIZING OPEN FUSION PORES

BUSTOS, MATÍAS; QUEVEDO, FLORENCIA; LUCCHESI, ORNELLA; MAYORGA, LUIS S; TOMES, CLAUDIA

Cairns, Queensland.

Congreso; The 18th International Symposium on Chromaffin Cell Biology; 2015

ISCCB

Sperm contain a single, large dense-core secretory granule (the acrosome) whose contents are secreted at fertilization by a special regulated exocytosis termed the acrosome reaction (AR). Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It was assumed that immediately afterwards fusion pores dilated spontaneously, originating tubules and vesicles that are shed, together with the acrosomal contents in the vicinity of the egg. The AR relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. We have conducted 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. Full length, geranylgeranylated and active Rab3A elicits human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins we demonstrated that the carboxy-terminus domain of the Rab3A molecule was necessary and sufficient to promote exocytosis whereas its amino-terminus 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 Rab3A?s inability to hydrolyze GTP. Under these conditions, SNARE proteins were engaged in botulinum toxin B-resistant- and α-SNAP/NSF-sensitive complexes; thus, we hypothesized that they had reached a post-fusion cis configuration. Because a dye applied to the medium entered the acrosome, we deduced that there was a connection between the intravesicular and extracellular compartments, most likely through open fusion pores. Yet, exocytosis was inhibited. Thus, we propose that inhibitory Rabs interfere with the vesiculation of membranes and release of the acrosomal contents after the opening of fusion pores.