Dumermuth E, Moore H P
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, 94720-3200, USA.
Methods. 1998 Oct;16(2):188-97. doi: 10.1006/meth.1998.0666.
To study biosynthetic transport through the constitutive and regulated secretory pathways, we have designed a semi-intact mammalian cell system that restores the transport of secretory proteins from the trans-Golgi/trans-Golgi network (TGN) to the cell surface. The mouse pituitary AtT-20 cell line is a suitable model to biochemically analyze molecular sorting in the secretory pathway. The prohormone proopiomelanocortin is sulfated on N-linked carbohydrate chains in the trans-Golgi prior to proteolytic processing in the secretory granule. Radiolabeling with [35S]sulfate therefore provides a convenient tool to selectively follow molecular events in the regulated secretory pathway without interference from earlier steps. Likewise, transport through the constitutive secretory pathway may be monitored using sulfate-labeled glycosaminoglycan chains. We show that export from the TGN is efficiently reconstituted in cells made semi-intact with streptolysin O, and is dependent on temperature, ATP and GTP hydrolysis, and cytosol. Packaging of proopiomelanocortin into immature secretory granules also activates the proteolytic processing machinery which eventually converts the prohormone to its bioactive mature product, adrenocorticotropic hormone. In addition, a large fraction of incompletely processed proopiomelanocortin is secreted as the processing intermediates from immature secretory granules. This process of constitutive-like secretion can be clearly distinguished from direct constitutive secretion from the trans-Golgi network by kinetic and compositional criteria. Furthermore, we have found that specific inhibitors of different protein phosphatases and kinases are potent blockers of constitutive and constitutive-like secretion. This experimental model should provide a valuable system to elucidate the molecular mechanism regulating post-Golgi traffic during secretory granule biogenesis.
为了研究通过组成型和调节型分泌途径的生物合成运输，我们设计了一种半完整的哺乳动物细胞系统，该系统可恢复分泌蛋白从反式高尔基体/反式高尔基体网络（TGN）到细胞表面的运输。小鼠垂体AtT-20细胞系是生化分析分泌途径中分子分选的合适模型。前激素阿黑皮素原在分泌颗粒中进行蛋白水解加工之前，先在反式高尔基体中的N-连接碳水化合物链上进行硫酸化。因此，用[35S]硫酸盐进行放射性标记提供了一种方便的工具，可在不受早期步骤干扰的情况下，选择性地追踪调节型分泌途径中的分子事件。同样，通过组成型分泌途径的运输可以使用硫酸盐标记的糖胺聚糖链进行监测。我们表明，在经链球菌溶血素O处理而变得半完整的细胞中，TGN的输出得到了有效重建，并且依赖于温度、ATP和GTP水解以及胞质溶胶。阿黑皮素原包装到未成熟分泌颗粒中也会激活蛋白水解加工机制，最终将前激素转化为其生物活性成熟产物促肾上腺皮质激素。此外，很大一部分未完全加工的阿黑皮素原作为加工中间体从未成熟分泌颗粒中分泌出来。这种组成型样分泌过程可以通过动力学和组成标准与反式高尔基体网络的直接组成型分泌清楚地区分开来。此外，我们发现不同蛋白磷酸酶和激酶的特异性抑制剂是组成型和组成型样分泌的有效阻滞剂。这个实验模型应该为阐明分泌颗粒生物发生过程中调节高尔基体后运输的分子机制提供一个有价值的系统。
