Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins.
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 GH(4)C(1) cells. 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 GH(4)C(1) cells 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 GH(4)C(1) cells, 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.
Department of Biological and Biophysical Sciences, University of Louisville Health Sciences Center, Louisville, Kentucky 40292, USA.
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SU
Gorr
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Huang
D J
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Cowley
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Kuliawat
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Arvan
D000818:Animals; D002460:Cell Line / Q000187:drug effects / Q000378:metabolism; D002864:Chromogranins / Q000737:chemistry* / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D011384:Proinsulin / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats
D002864:Chromogranins; D004220:Disulfides; D007328:Insulin; D011384:Proinsulin; D004229:Dithiothreitol