Gorr S U, Huang X F, Cowley D J, Kuliawat R, Arvan P
Department of Biological and Biophysical Sciences, University of Louisville Health Sciences Center, Louisville, Kentucky 40292, USA.
Am J Physiol. 1999 Jul;277(1):C121-31. doi: 10.1152/ajpcell.1999.277.1.C121.
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.
对于几种分泌蛋白，有人提出二硫键连接的环结构是分选至分泌颗粒所必需的。为了探究这一假说，我们在活的胰岛以及PC-12和GH(4)C(1)细胞中采用了二硫苏糖醇（DTT）处理。在胰岛中，远端分泌途径中的二硫键还原并未增加胰岛素原（或胰岛素）的组成型或组成型样分泌。在PC-12细胞中，DTT处理导致新合成的嗜铬粒蛋白B（CgB）在未受刺激时的分泌显著增加，这可能是由于还原了单个保守的嗜铬粒蛋白二硫键（E. 查纳特、U. 魏斯、W. B. 胡特纳和S. A. 图兹。《欧洲分子生物学组织杂志》12: 2159 - 2168, 1993）。然而，在同样内源性合成CgB的GH(4)C(1)细胞中，DTT处理减少了新合成的催乳素并阻断了其输出，而新合成的CgB则正常地被转运至分泌颗粒。此外，通过刺激分泌测定法检测，在GH(4)C(1)细胞中瞬时表达时，CgA和缺乏保守二硫键的CgA突变体表现出可比的多聚体聚集特性以及靶向分泌颗粒的能力。因此，由二硫键破坏引起的调节性分泌蛋白的构象扰动导致了在蛋白质转运方面既依赖于蛋白质又依赖于细胞类型的后果。
