PRA1 and synaptotagmin VIII: Regulators of vesicle trafficking and fusion

Abstract

The work described within my doctoral thesis examines the localization and functional role of the prenylated Rab acceptor 1 (PRA1) in the Rab GTPase cycle and the expression, localization and functional role of synaptotagmin VIII in the mouse sperm acrosome reaction. My work began with the examination of both PRA1 and synaptotagmin VIII. The PRA1 project was taken over by other graduate students while my interests remained focused on elucidating the role of synaptotagmin VIII in sperm. Rab is a family of small Ras-like GTPases regulating intracellular vesicle transport. Prenylated Rab Acceptor (PRA1) has previously been shown to interact with Rab GTPases and VAMP2. I have examined the subcellular localization of PRA1 and examined the functional significance of its interaction with Rab3A. PRA1 is localized to the Golgi complex while truncation of the carboxy-terminus results in a mislocalization to the ER. I also observed that PRA1 is able to bind weakly to GDP dissociation inhibitor (GDI), a protein involved in the solubilization of membrane-bound Rab GTPases. Furthermore, the addition of PRA1 inhibited the extraction of membrane-bound Rab3A by GDI, suggesting that membrane localization of Rab GTPases is dependent on the opposing action of PRA1 and GDI. The sperm acrosome is a large secretory granule that undergoes calcium-stimulated exocytosis by a mechanism analogous to neuronal secretion. In neurons, the core SNARE complex, composed of syntaxin (Stx), SNAP-25 and VAMP2, mediates vesicle fusion, while calcium regulation is thought to be accomplished by the synaptotagmin (Syt) family via calcium-dependent binding to syntaxin and SNAP-25. I examined the expression, localization and function of Syt VIII in mouse sperm. I found that Syt VIII is expressed in spermatozoa and localizes to the acrosome. In addition, the introduction of Syt VIII antibodies and recombinant protein into Streptolysin O permeabilized sperm, resulted in inhibition of the acrosome reaction (AR), suggesting a critical function for this protein. Syt VIII also exhibited Ca2+-dependent binding to Stx2 with an EC50 that closely matches the calcium requirement of the AR. Taken together, our data suggest a critical role for Syt VIII and Stx2 in the acrosome reaction.