Chanat E, Weiss U, Huttner W B, Tooze S A
Institute for Neurobiology, University of Heidelberg, Germany.
EMBO J. 1993 May;12(5):2159-68. doi: 10.1002/j.1460-2075.1993.tb05864.x.
The role of the single, highly conserved disulfide bond in chromogranin B (secretogranin I) on the sorting of this regulated secretory protein to secretory granules was investigated in the neuroendocrine cell line PC12. Treatment of PC12 cells with dithiothreitol (DTT), a membrane permeable thiol reducing agent known to prevent disulfide bond formation in intact cells, resulted in the secretion of newly synthesized chromogranin B, but only slightly decreased the intracellular storage of newly synthesized secretogranin II, a regulated secretory protein devoid of cysteines. The secretion of newly synthesized chromogranin B in the presence of DTT occurred with similar kinetics to those of a heparan sulfate proteoglycan, a known marker of the constitutive secretory pathway in PC12 cells. Analysis of the various secretory vesicles derived from the trans-Golgi network (TGN) indicated that DTT treatment diverted newly synthesized chromogranin B to constitutive secretory vesicles, whereas the packaging of secretogranin II into immature secretory granules was unaffected by the reducing agent. The chromogranin B molecules diverted to constitutive secretory vesicles, in contrast to those stored in secretory granules, were found to contain free sulfhydryl residues. The effect of DTT on chromogranin B occurred in the TGN rather than in the endoplasmic reticulum. We conclude that the sorting of CgB in the TGN to secretory granules is dependent upon the integrity of its single disulfide bond.
在神经内分泌细胞系PC12中，研究了嗜铬粒蛋白B（分泌粒蛋白I）中单一高度保守的二硫键在这种受调节的分泌蛋白分选至分泌颗粒过程中的作用。用二硫苏糖醇（DTT）处理PC12细胞，DTT是一种已知可防止完整细胞中二硫键形成的膜通透性巯基还原剂，结果导致新合成的嗜铬粒蛋白B分泌，但仅略微降低了新合成的分泌粒蛋白II（一种不含半胱氨酸的受调节分泌蛋白）的细胞内储存。在DTT存在下新合成的嗜铬粒蛋白B的分泌动力学与硫酸乙酰肝素蛋白聚糖（PC12细胞组成型分泌途径的已知标志物）相似。对源自反式高尔基体网络（TGN）的各种分泌小泡的分析表明，DTT处理使新合成的嗜铬粒蛋白B转向组成型分泌小泡，而分泌粒蛋白II包装到未成熟分泌颗粒中不受还原剂影响。与储存在分泌颗粒中的嗜铬粒蛋白B分子相比，转向组成型分泌小泡的嗜铬粒蛋白B分子含有游离巯基残基。DTT对嗜铬粒蛋白B的作用发生在TGN而非内质网中。我们得出结论，TGN中嗜铬粒蛋白B分选至分泌颗粒取决于其单一二硫键的完整性。
