Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic beta cells and its physiological function in exo-endocytosis of secretory granules

Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic beta cells and its implication in the exo-endocytosis of insulin and gamma -amino butyric acid (GABA). Immunocytochemistry showed that endo brevin is associated with tubulo-vesicular structures, which are colocalize d with early endosomes labeled by early endosome antigen (EEA)-1 in insulin oma MIN6 cells, To determine the cellular localization of endobrevin, we ap pended the green fluorescent protein (GFP) to endobrevin and the fusion pro tein was introduced into MIN6 cells. The subcellular localization of GFP-en dobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP-endobrevin and endocytosed Texas-Red(Tx-R) label ed transferrin receptor and immunocytochemistry with anti-EEA1 antibody rev ealed that endobrevin was preferentially localized in the early endosome. T hen, we examined the functional role of endobrevin in the exocytosis of ins ulin and GABA from pancreatic beta cells. Endobrevin overexpression increas ed the amount of GABA released from MIN6 cells; in contrast, it decreased t he glucose-stimulated insulin release from rat islets, MIN6 and INS1-D cell s to approximately 50% of the control levels. Both in vitro and in vivo bin ding studies showed that endobrevin binds to syntaxin 1, Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression a ccelerates vesicle recycling, We conclude that (1) endobrevin is localized in the early endosome in pancreatic beta cells and (2) endobrevin plays a p hysiological role in the exo-endocytosis of insulin and GABA from pancreati c beta cells, probably via an interaction between endocytic vesicles and th e endosome.