title: Role of H+-ATPase-mediated acidification in sorting and release of the regulated secretory protein chromogranin A: evidence for a vesiculogenic function.
abstract: The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells. Selective aggregation of secretory granule constituents in an acidic, bivalent cation-rich environment is considered to be a prerequisite for sorting to the regulated secretory pathway. The effect of selective vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 on the pH gradient along the secretory pathway was used here to study the role of acidification on the trafficking of the regulated secretory protein chromogranin A (CgA) in PC12 cells. Sorting of CgA was assessed by three-dimensional deconvolution microscopy, subcellular fractionation, and secretagogue-stimulated release, examining a series of full-length or truncated domains of human CgA (CgA-(1-115), CgA-(233-439)) fused to either green fluorescent protein or to a novel form of secreted embryonic alkaline phosphatase (EAP). We show that a full-length CgA/EAP chimera is sorted to chromaffin granules for exocytosis. Inhibition of V-ATPase by bafilomycin A1 markedly reduced the secretagogue-stimulated release of CgA-EAP by perturbing sorting of the chimera (at the trans-Golgi network or immature secretory granule) rather than the late steps of exocytosis. The effect of bafilomycin A1 on CgA secretion depends on a sorting determinant located within the amino terminus (CgA-(1-115)) but not the C-terminal region of the granin. Moreover, examination of chromaffin granule abundance in PC12 cells exposed to bafilomycin A1 reveals a substantial decrease in the number of dense-core vesicles. We propose that a V-ATPase-mediated pH gradient in the secretory pathway is an important factor for the formation of dense-core granules by regulating the ability of CgA to form aggregates, a crucial step that may underlie the granulogenic function of the protein.
authors.affiliation: Department of Medicine and Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093, USA. ltaupenot@ucsd.edu
authors.forename: Laurent
authors.initials: L
authors.lastname: Taupenot
authors.forename: Kimberly L
authors.initials: KL
authors.lastname: Harper
authors.forename: Daniel T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D000143:Acids / Q000378:metabolism*; D000468:Alkalies / Q000378:metabolism; D000818:Animals; D001679:Biogenic Amines / Q000378:metabolism; D053379:Chromogranin A; D002864:Chromogranins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008163:Luminescent Measurements; D018942:Macrolides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008985:Monensin / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D016716:PC12 Cells; D021381:Protein Transport / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000143:Acids; D000468:Alkalies; D001679:Biogenic Amines; C506150:CHGA protein, human; D053379:Chromogranin A; D002864:Chromogranins; D004791:Enzyme Inhibitors; D007476:Ionophores; D018942:Macrolides; D011993:Recombinant Fusion Proteins; C506153:chromogranin A, rat; C040929:bafilomycin A1; D008985:Monensin; D006180:Proton-Translocating ATPases; D009550:Nigericin