RESUMO
At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. 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 is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction.
Assuntos
Exocitose , Espermatozoides/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Acrossomo/metabolismo , Cálcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Masculino , Proteínas rab de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/genéticaRESUMO
The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.
Assuntos
Exocitose/fisiologia , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas SNARE/metabolismo , Espermatozoides/fisiologia , Reação Acrossômica/fisiologia , Animais , Cálcio/metabolismo , Humanos , Masculino , Proteínas Sensíveis a N-Etilmaleimida/genética , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE/genética , Espermatozoides/citologia , Tirosina/metabolismo , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.