Cool D R, Louie D Y, Loh Y P
Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Endocrinology. 1996 Dec;137(12):5441-6. doi: 10.1210/endo.137.12.8940369.
The subcellular localization and functionality of transfected yeast aspartic protease 3 (YAP3p) in a mammalian cell line were investigated. The complementary DNAs encoding the prohormone-processing enzyme (YAP3p) and a prohormone, bovine POMC, were cotransfected into PC12 (rat pheochromocytoma) cells. Immunocytochemical analysis of the cells using a YAP3p antibody showed a perinuclear punctate distribution of YAP3p in the cell body as well as immunostaining in the tips of the neurites. This pattern of immunostaining indicates localization of YAP3p in secretory granules. Analysis of the processing of POMC showed that in cells transfected with the POMC complementary DNA alone, only POMC was found, indicating a lack of processing of the prohormone. However, in cells coexpressing YAP3p, the POMC was completely processed to yield ACTH-(1-39) and ACTH-(1-14), consistent with the specificity of YAP3p found in vitro. Pulse-chase studies showed that POMC was processed after 20 min of chase, suggesting that processing occurred in the late Golgi network and continued in the secretory granules. Western blot analysis determined that YAP3p was secreted from the cells in a regulated manner. This study provides the first demonstration that a yeast prohormone-processing enzyme (YAP3p) of the aspartic protease class can be sorted correctly to secretory granules and activated to process a prohormone (POMC) in a highly efficient manner in mammalian cells.
研究了转染的酵母天冬氨酸蛋白酶3（YAP3p）在哺乳动物细胞系中的亚细胞定位和功能。将编码激素原加工酶（YAP3p）和一种激素原——牛阿黑皮素原（POMC）的互补DNA共转染到PC12（大鼠嗜铬细胞瘤）细胞中。使用YAP3p抗体对细胞进行免疫细胞化学分析，结果显示YAP3p在细胞体中呈核周点状分布，并且在神经突尖端有免疫染色。这种免疫染色模式表明YAP3p定位于分泌颗粒中。对POMC加工过程的分析表明，在仅转染POMC互补DNA的细胞中，仅发现了POMC，这表明激素原缺乏加工。然而，在共表达YAP3p的细胞中，POMC被完全加工产生促肾上腺皮质激素（ACTH）-（1-39）和促肾上腺皮质激素（ACTH）-（1-14），这与体外发现的YAP3p的特异性一致。脉冲追踪研究表明，在追踪20分钟后POMC被加工，这表明加工发生在晚期高尔基体网络中，并在分泌颗粒中持续进行。蛋白质印迹分析确定YAP3p以调节的方式从细胞中分泌。这项研究首次证明，天冬氨酸蛋白酶类的酵母激素原加工酶（YAP3p）可以正确分选到分泌颗粒中，并在哺乳动物细胞中高效激活以加工激素原（POMC）。
