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

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.The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells (1). The constitutive pathway allows rapid transport of proteins in small vesicles originating from the outermost layer of the Golgi complex (the trans-Golgi network, or TGN 1 ) and destined for rapid fusion with the plasma membrane. The regulated secretory pathway is characterized by the concentration of a selected (segregated) pool of secretory proteins into granules with a typical electron-dense appearance on transmission electron microscopy. These granules may remain in the cell for an extended period of time after their formation until prompted to undergo exocytotic fusion with the plasma membrane by a secretagogue characteristic for a particular cell type.Selective aggregation or condensation of secretory granule constituents in a mildly acidic environment and in the presence of millimolar concentrations of bivalent cations like Ca 2ϩ is considered to be an important prerequisite for two proposed models of sorting within the regulated secretory pathway, namely sorting-for-entry and sorting-by-retention (2-4). In the sorting-for-entry model, th...

Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells

“…(Fig. 1C), consistent with DCG abundance in naive PC12 cells (37). In contrast, the number of DCGs was diminished by ϳ44% (to 0.40 Ϯ 0.04 DCG/m 2 ) in response to ϳ75% reduction of SgII expression (Fig.…”

“…Sucrose Gradient Fractionation-A postnuclear supernatant from homogenized cells was prepared and subjected to sucrose density gradient fractionation (0.6 -2.2 M) as described previously (37). Fractions were collected and assayed for sucrose concentration by refractometry and immunoblotted for HA and CgB immunoreactivity after trichloroacetic acid precipitation.…”

“…3A), typical behavior of constitutively secreted proteins in sympathoadrenal cells, including A35C cells (supplemental Fig. S2A) (10,23,28,37). In sharp contrast, SgII-GFP, SgII-EAP, or SgII-HA displayed a highly punctate distribution throughout the cell body (Fig.…”

“…2, A and B), likely resulting from the use of an alternative/internal translation initiation (AUG) site reported to function in mitochondrial targeting of NPY in non-neuroendocrine or secretorydeficient neuroendocrine cells (42)(43)(44)(45); and (ii) a juxtanuclear distribution, co-localizing with the TGN-resident glycoprotein ␤-1,4 galactosyltransferase (GalT-CFP) (1.2% of transfected cells; R p ϭ 0.67 Ϯ 0.03; R o ϭ 0.67 Ϯ 0.03; 8 cells, n ϭ 124 optical sections; Fig. 2, A and B), reflecting the incomplete sorting of NPY-GFP at the TGN (37).…”

“…S2C) (23,37,50). In COS-7 cells, the release of the constitutive secretory protein SIG-EAP was elevated in basal, nonstimulating conditions (7.8 Ϯ 0.6%; Fig.…”