INTRACELLULAR TRAFFICKING AND ACTIVATION OF THE FURIN PROPROTEIN CONVERTASE - LOCALIZATION TO THE TGN AND RECYCLING FROM THE CELL-SURFACE
INTRACELLULAR TRAFFICKING AND ACTIVATION OF THE FURIN PROPROTEIN CONVERTASE - LOCALIZATION TO THE TGN AND RECYCLING FROM THE CELL-SURFACE
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DOI：
10.1002/j.1460-2075.1994.tb06231.x
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发表时间：
1994-01-01
期刊：
影响因子：
11.4
通讯作者：
THOMAS, G
中科院分区：
文献类型：
--
作者：
MOLLOY, SS;THOMAS, L;THOMAS, G
弗林蛋白酶是一种膜相关的内切蛋白酶，其有效地切割在共有序列Arg-X-Lys/Arg-Arg的C-末端侧上的前体蛋白，并且已经被提出在胞吐和胞吞隔室中催化这些反应。为了研究其生物合成和路由，使用含有插入在酶的自动蛋白水解成熟位点的C-末端侧的FLAG表位标签的弗林蛋白酶构建体（指定为fur/f）。表位标签的引入对弗林蛋白酶的表达、蛋白水解成熟或活性没有影响。通过免疫荧光显微镜对Fur/f的定位分析表明，其染色模式与几种高尔基体相关标记物的染色模式在很大程度上重叠。用布雷菲德菌素A处理细胞导致毛/f分布在微管组织中心周围崩溃，表明弗林蛋白酶集中在高尔基体网络（TGN）中。免疫电子显微镜显示，明确弗林蛋白酶驻留在TGN共定位与TGN 38。与其在多个隔室中的拟议活性一致，抗体摄取研究表明，细胞表面和TGN之间的fur/f循环。此外，靶向TGN需要酶的胞质尾区中的序列。脉冲追踪和免疫荧光分析表明，前区去除发生在内质网和切割可能需要退出这个区室。最后，我们表明，proregion去除是必要的，但不足以激活酶。
Furin is a membrane-associated endoprotease that efficiently cleaves precursor proteins on the C-terminal side of the consensus sequence, Arg-X-Lys/Arg-Arg, and has been proposed to catalyze these reactions in both exocytic and endocytic compartments. To study its biosynthesis and routing, a furin construct (designated fur/f) containing the FLAG epitope tag inserted on the C-terminal side of the enzyme's autoproteolytic maturation site was used. Introduction of the epitope tag had no effect on the expression, proteolytic maturation or activity of furin. Analysis of the localization of fur/f by immunofluorescence microscopy showed that its staining pattern largely overlapped with those of several Golgi-associated markers. Treatment of cells with brefeldin A caused the fur/f distribution to collapse around the microtubule organizing center, indicating that furin is concentrated in the trans-Golgi network (TGN). Immunoelectron microscopy showed unequivocally that furin resides in the TGN where it colocalized with TGN38. In agreement with its proposed activity in multiple compartments, antibody uptake studies showed that fur/f cycles between the cell surface and TGN. Furthermore, targeting to the TGN requires sequences in the cytoplasmic tail of the enzyme. Pulse- chase and immunofluorescence analyses demonstrated that proregion removal occurs in the endoplasmic reticulum and that cleavage may be required for exit from this compartment. Finally, we show that proregion removal is necessary but not sufficient for enzyme activation.