UCSF

The regulated secretion of peptide hormones, neuropeptides and many growth factors depends on their sorting into large dense-core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used RNAi to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal pathway, and the contribution of dysregulated peptide hormone secretion has not been investigated. We now find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble and membrane LDCV cargo, reducing the extent of stimulation. We also observe increased basal release of insulin and glucagon from AP-3-deficient pancreatic islet cells, suggesting a widespread disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but we find that loss of both is required to impair LDCV production. In addition, we show that loss of the related adaptor protein AP-1 has no effect on regulated secretion but greatly exacerbates the effect of AP-3 RNAi, indicating distinct roles for the two adaptors in formation of the regulated secretory pathway. Finally, quantitative proteomic analysis of LDCVs reveals a number of functionally important membrane proteins whose levels are significantly altered by the loss of AP-3. In particular, ectopic localization of the t-SNARE, syntaxin-1A, to LDCVs may account for the impairment in regulated secretion observed after loss of AP-3. Taken together, these results support the notion that AP-3-directed sorting maintains the normal complement of LDCV membrane proteins, thus ensuring the proper regulation of secretion.