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J. Biol. Chem., Vol. 280, Issue 5, 3885-3897, February 4, 2005

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Role of H⁺-ATPase-mediated Acidification in Sorting and Release of the Regulated Secretory Protein Chromogranin A

EVIDENCE FOR A VESICULOGENIC FUNCTION^*

Laurent Taupenot¶, Kimberly L. Harper, and Daniel T. O'Connor||

From the Department of Medicine and ^||Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093 and Veterans Affairs San Diego Healthcare System, San Diego, California 92161

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.

Received for publication, July 20, 2004 , and in revised form, November 1, 2004.

^* This work was supported by NIDDK, National Institutes of Health Grant DK59628 (to L. T.), by the Medicine Education and Research Foundation (to L. T.), and by the National Institutes of Health and the Department of Veterans Affairs (to D. T. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: University of California at San Diego, Dept. of Medicine (0838), 9500 Gilman Dr., La Jolla CA 92093-0838. Tel.: 858-534-0670; Fax: 858-534-0626; E-mail: ltaupenot{at}ucsd.edu.

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