Permeable GTP-bound Rab27A exhibits a positive role during human sperm dense-core granule exocytosis

LUCCHESI, ORNELLA; TOMES, CLAUDIA; BUSTOS, MATÍAS

La Serena

Workshop; EMBO Workshop: Actualizations in membrane trafficking in health and disease; 2016

EMBO

Exocytosis of sperm´s single secretory granule or acrosome (acrosome reaction, AR)is a highly regulated event essential for fertilization. The small G proteins Rabs 3 and 27 control the activation cascade that allows SNAREs assembly for membrane fusion during AR. Because sperm are transcriptionally and translationally inactive, overexpression or silencing experiments cannot be performed. An alternative method is the use of membrane-permeant proteins. We have engineered a permeable version of Rab27A by introducing the sequence encoding the TAT peptide of HIV virus fused to the N-terminal end of Rab27A in a bacterial expression plasmid. We expressed the protein fused to His6 in Escherichia coli, geranylgeranylated it or not in vitro, and loaded it with GDP-β-S or GTP-ɣ-S. To analyze the incorporation of permeable Rab27A into sperm and its effect on the signaling events involved in the AR, we incubated cells with different versions of the protein and quantified the AR. Rab27A geranylgeranylated and loaded with GTP-ɣ-S transduced into the cells and stimulated the AR. The others versions of Rab27A did not affect exocytosis per se but prevented the progesterone or 8-pCPT-2?-O-Me-cAMP-triggered AR. These preliminary results suggest that Rab27A must be active and associated to membranes to accomplish its regulatory function. By a method that combines protein-protein interaction with indirect immunofluorescence designed to detect small G proteins in their active state, we analyzed the effect of permeant Rab27A on the activation status of other small GTPases required for the AR. Rab27A, in its prenylated and active state, triggers the activation of Rap1 and Rab3A, a process required for calcium release from the acrosome and membrane SNARE-dependent fusion. Our results indicate that the activation of Rab27A is an essential step that allows the downstream activation of others components of the signaling cascade involved in sperm dense-core granule exocytosis.