α-SNAP and NSF are required in a priming step during the human sperm acrosome reaction

Abstract

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.

A Novel Site of Action for α-SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion

“…These findings reveal a second and unexpected site of action of ␣-SNAP on the SNARE assembly-disassembly cycle, and they provide a molecular explanation for the inhibition of membrane fusion by ␣-SNAP observed in previous studies (Babcock et al, 2004;Tomes et al, 2005).…”

“…Increasing ␣-SNAP dosage in Drosophila resulted in diminished neuronal exocytosis, accumulation of SNARE complexes, and other morphological defects (Babcock et al, 2004). Furthermore, addition of ␣-SNAP was shown to inhibit cell-free fusion reactions, including sperm acrosome exocytosis (Tomes et al, 2005) and yeast vacuole fusion (Wang et al, 2000). In all cases, concomitant increase in NSF levels was able to overcome the inhibition, but the mechanism of ␣-SNAP-mediated inhibition was not identified.…”

Sustained High Protein-tyrosine Phosphatase 1B Activity in the Sperm of Obese Males Impairs the Sperm Acrosome Reaction

“…After progesterone stimulation, PTP1B and NSF are co-localized in the acrosomal region. In addition, ␣-SNAP, an adaptor protein that mediates the binding of NSF to SNARE complexes, has been proved to be mainly in the cytosol of capacitated mouse spermatozoa (34), which control the initiation of AR before progesterone treatment. Taken together, AR is a tightly regulated process by segmentation of multiple key proteins to avoid spontaneous reaction.…”

CaMKIIα interacts with multi-PDZ domain protein MUPP1 in spermatozoa and prevents spontaneous acrosomal exocytosis

“…One of these characteristics is the formation of hundreds of fusion points between the plasma membrane and the outer acrosomal membrane, ensuring the efficient delivery of the entire acrosomal contents (Barros et al, 1967;Michaut et al, 2000;Zanetti and Mayorga, 2009). Although there are currently no experimental data to explain this special feature of synchronous membrane pore formation during acrosomal secretion, an intriguing solution for this conundrum could be a scenario in which distinct pre-assembled SNARE complexes Roggero et al, 2007;Tomes et al, 2005;Zarelli et al, 2009) are mechanically coupled in a functional protein network between the outer acrosomal membrane and the adjacent plasma membrane. However, if mechanically coupled SNARE complexes are the basis of the observed synchronized fusion pore formation, a single 'unintended' fusion event could be able to trigger a 'domino' effect.…”