DETECTION OF G-PROTEIN HETEROTRIMERS ON LARGE DENSE CORE AND SMALL SYNAPTIC VESICLES OF NEUROENDOCRINE AND NEURONAL CELLS

Heterotrimeric G proteins, initially believed to be exclusively presen t in the plasma membrane, have also been found to be associated with i ntracellular membrane compartments. There they are involved in various membrane trafficking processes including regulated secretion (reviewe d in Bomsel, M., K. Mostov, Mol. Biol. Cell 3, 1317-1328 (1999)). Vesi cles of two distinct types enter the regulated secretory pathway, i.e. large dense core vesicles and small synaptic vesicles, which differ i n their membrane composition and content. Little is known about an ass ociation of heterotrimeric G proteins,vith regulated secretory vesicle s, that would explain some aspects of the role heterotrimeric G protei ns have during secretion. By immunofluorescence microscopy and immunor eplica analysis, we provide the first demonstration of the presence of complete sets of heterotrimeric G proteins, consisting of alpha-, bet a-, and gamma-subunits, on large dense core vesicles from bovine adren al medulla (chromaffin granules) and small synaptic vesicles from rode nt and bovine brain. Each of the two types of secretory vesicles conta ins beta-subunits (at least beta(1) and beta(2)), as well as gamma-sub units (at least gamma(2) or gamma(3). Interestingly, they differ in th eir composition of alpha-subunits. On small synaptic vesicles, we foun d two G(o) alpha-subunits (alpha(o1)) and alpha(o2)) and two G(i) alph a-submits (alpha(i1) and alpha(i2)). In contrast, on chromaffin granul es so far only one alpha(o)-subunit but no alpha(i)-subunits could be detected. Functional properties such as transmitter storage and/or exo cytotic membrane fusion mag be modulated by the various G-protein subu nits associated with chromaffin granules and small synaptic vesicles.