Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins

Abstract

For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH4C1cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J.12: 2159–2168, 1993). However, in GH4C1cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH4C1cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.

N- and C-terminal Domains Direct Cell Type-specific Sorting of Chromogranin A to Secretory Granules

“…In contrast, it is shown that the C-terminal domain of CgA is not necessary for sorting to the regulated secretory pathway in PC12 cells. Together with previous results (11), these findings show that CgA contains two independent cell-specific sorting domains.…”

“…The cells were plated in culture medium at 5 ϫ 10 5 cells/well in six-well culture plates (5 ϫ 10 4 cells/cm 2 ). Cloning and Mutagenesis-The expression plasmids for wild-type bovine CgA, CgA⌬CC (missing the disulfide bond), and secreted alkaline phosphatase (SEAP) were described previously (11,23). CgA341 and CgA387 were prepared from wild-type CgA by site-directed mutagenesis with primers from Genosys Biotechnologies Inc. (The Woodlands, TX).…”

“…Transient Expression of CgA in GH4C1 or PC12 Cells-Transfections of GH4C1 and PC12 cells were performed as described previously (11). The pcDNA3/CgA, pcDNA3/CgA⌬CC, pcDNA3/CgA341, pcDNA3/ CgA387, pcDNA3/SEAP plasmids (and pcDNA3 as a control) were used to transiently transfect GH4C1 or PC12 cells in separate experiments.…”

“…18). However, we have recently found that the N-terminal disulfide bond that is necessary for sorting of chromogranin B in neuroendocrine PC12 cells (14) is not required for sorting in endocrine GH4C1 cells (11). This result suggests that, in endocrine cells, the signals and mechanisms used for sorting of regulated secretory proteins into secretory granules are different from those used in neuroendocrine cells.…”

“…Thus, pro-opiomelanocortin (POMC) 1 is sorted by binding to carboxypeptidase E and this sorting depends on an N-terminal disulfide bridge in POMC (9). Chromogranins contain a similar N-terminal disulfide bond that is both necessary and sufficient for sorting in PC12 cells (11)(12)(13)(14), although carboxypeptidase E does not appear to mediate this sorting (11,15). While chromogranins aggregate at low pH and in the presence of calcium, this aggregation is neither necessary nor sufficient for sorting in PC12 cells (12,16).…”