G(I) REGULATION OF SECRETORY VESICLE SWELLING EXAMINED BY ATOMIC-FORCE MICROSCOPY

In the last decade, several monomeric and heterotrimeric guanine nucle otide binding proteins have been identified to associate with secretor y vesicles and to be implicated in exocytosis, Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion a t the plasma membrane, However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secr etory vesicle volume in the exocytotic process remains unclear, In thi s study, we report association of the secretory vesicle membrane with the alpha subunit of a heterotrimeric GTP binding protein G(alpha i3) and implicate its involvement in vesicle swelling, Using an atomic for ce microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicl es in exocrine pancreas, Exposure of zymogen granules to GTP resulted in a 15-25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined b y confocal microscopy, Mas7, an active mastoparan analog known to stim ulate G(i) proteins, was found to stimulate the GTPase activity of iso lated zymogen granules and cause swelling, Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitiv e, Ca2+ had no effect on zymogen granule size, Taken together, the res ults indicate that G(alpha i3) protein localized in the secretory vesi cle membrane mediates vesicle swelling, a potentially important prereq uisite for vesicle fusion at the cell plasma membrane.