Rab3 is present on endosomes from bovine chromaffin cells in primary culture

Rab3a, a small GTP-binding protein, is believed to mediate Ca2+-dependent e xocytosis, Consistent with such a role was the previously reported specific association of Rab3a with synaptic vesicles in neurons and secretory granu les in adrenal chromaffin cells. Secretory vesicles are believed to be the final point of Rab3a membrane association, as it was shown by several group s that Rab3a dissociates from the secretory vesicle membrane during stimula ted exocytosis. In chromaffin cells, Rab3a is not exclusively localized on secretory granules since a fraction is present on a previously unidentified subcellular compartment equilibrating at light sucrose density. This 'ligh t' membraneous structure could be the starting point for reassociation of R ab3a with membranes involved in granule formation, or it could be a structu re unrelated to granules. The present study used several subcellular fractionation techniques and imm unomicroscopy to unravel the nature of the 'light' Rab3a-containing structu res from bovine chromaffin cells in primary culture. After stimulation, amo unts of both Rab3a-d and the granule marker dopamine-beta-hydroxylase (D be ta H) increase transiently in sucrose gradient fractions enriched in endoso mal markers. A diaminobenzidine-induced density shift of endosomes alters t he distribution of D beta H and Rab3a-d. At the ultrastructural level, subp lasmalemmal pleiomorphic organelles were detected by Rab3a-d-immunogold lab elling, Taken together our data provide for the first time evidence that internalis ed secretory granule membranes go through an endosomal stage where Rab3a is present, resembling the neuronal synaptic vesicle cycle. This indicates th at the endosome is an important trafficking route in the biogenesis/recycli ng of secretory vesicles in chromaffin cells, in which Rab3a could have an as yet unknown regulatory function, and could point to the existence of alt ernative recycling pathways for the chromaffin granule membrane.