THE GTPASE RAB3A IS ASSOCIATED WITH LARGE DENSE CORE VESICLES IN BOVINE CHROMAFFIN CELLS AND RAT PC12 CELLS

Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise sub cellular localization, It has been suggested that Rab3a, which is main ly expressed in neural and endocrine cells, might regulate exocytosis, Recently, direct experimental evidence supporting this hypothesis has been obtained, Consistent with such a role for Rab3a in regulated exo cytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin c ells, Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localizat ion of this GTP-binding protein by using a combination of morphologica l techniques, Bovine chromaffin cells were labelled with an affinity-p urified polyclonal anti-Rab3a antibody and analyzed by confocal microc opy, Rab3a was found to colocalize partially with dopamine beta-hydrox ylase, a chromaffin granule marker, In agreement with this observation , immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicl es were labelled, To eliminate the possibility that the staining was d ue to a Rab3a-reiated protein, we investigated by immunoelectron micro scopy the localization of an epitope-tagged Rab3a expressed in rat PC1 2 cells, Secretory granules were specifically labelled, whereas clear microvesicles were not, These results provide further evidence support ing a specific association of the GTPase Rab3a with large dense core s ecretory vesicles.