Gai3 in Pancreatic Zymogen Granules Participates in Vesicular Fusion1

Earlier studies indicated that a Gj-like protein localized in pancreatic zymogen granule (ZG) membrane mediates vesicle swelling, and is a potentially important prerequisite for vesicle fusion at the cell plasma membrane (PM) [Jena et al. (1997) Proc. NatL Acad. Set USA 94, 13317-13322]. In the present study, we demonstrate the presence of Gai3 immunoreactivity in ZGs of rat exocrine pancreas using immunoblot assays, light and electron immunomicroscopy. Since GTP has been implicated in the fusion of isolated ZG with PM fractions [Nadin et al. (1989) J. Cell Biol. 109, 2801-2808], the potential role of ZG-associated Gai3 was investigated. Immunoblot assays demonstrate an increase in Gai3 protein in ZGs isolated from carbamylcholine stimulated pancreas. Thin layer chro-matography shows an increase in GTP hydrolysis by GTPase in ZGs isolated from stimu-lated compared to resting pancreas. In vitro fusion assays demonstrate that ZGs isolated from carbamylcholine-stimulated pancreatic lobules fuse with the PM at a greater po-tency in the presence of GTP, mastoparan (G protein agonist) and its analogue mas7. Furthermore, Gai3-specific a recombinant GAIP (G alpha interacting protein), potenti-ates ZG-PM fusion in the presence of GTP but not in presence of the non-hydrolyzabl