Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis

The Rab27 effectors play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationship by comparing the exocytic phenotypes in beta cells simultaneously lacking two effectors with those lacking one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy indicate that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. Exophilin-8 also promotes fusion of granules stably docked to the plasma membrane mediated by granuphilin, although it is dispensable for granule residence beneath the plasma membrane. The current study presents the first diagram for multiple intracellular paths of granule exocytosis and for functional hierarchy among different Rab27 effectors in the same cell.