Cruz I P, Nillni E A
Division of Endocrinology, Department of Medicine, Brown University School of Medicine, Rhode Island Hospital, Providence, Rhode Island 02903, USA.
J Biol Chem. 1996 Sep 13;271(37):22736-45. doi: 10.1074/jbc.271.37.22736.
Post-translational processing of proteins plays a key role in regulating their subcellular localization, enzymatic activity, and protein-protein interactions by such diverse mechanisms as phosphorylation, glycosylation, and proteolytic cleavage. The prothyrotropin-releasing hormone (pro-TRH) precursor (26 kDa) undergoes proteolytic cleavage at either of two sites, generating a 15/10-kDa or a 9.5/16.5-kDa N/C-terminal pair of intermediates. Using transfected AtT20 cells encoding a prepro-TRH cDNA, we have previously reported that this initial set of cleavages occurs prior to entry into the secretory granules (Nillni, E. A., Sevarino, K. A., and Jackson, I. M. D. (1993) Endocrinology 132, 1271-1277). In this study, we set out to identify the subcellular compartment where this initial cleavage takes place as well as to determine the sites of processing of the intermediates produced. Our strategy was to block the transport of pro-TRH or its intermediates from one subcellular compartment to the next and to assay for the accumulation of intermediates, presumably because their processing occurs in a post-blockade compartment. Radiolabeling experiments in AtT20 cells in the presence of the drug brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi complex, led to an accumulation of the 26-kDa precursor, suggesting a post-endoplasmic reticulum site of processing. When Golgi complex-to-secretory granule transport was blocked at 20 degrees C, the processing of the 26-kDa precursor was not affected, suggesting a Golgi complex site of processing. At this temperature, the 15-kDa N-terminal intermediate accumulated, suggesting a post-Golgi complex processing site, while the 16.5-kDa C-terminal intermediate was processed in the Golgi complex to produce a 5.4-kDa peptide.
蛋白质的翻译后加工通过磷酸化、糖基化和蛋白水解切割等多种机制，在调节其亚细胞定位、酶活性和蛋白质 - 蛋白质相互作用方面发挥关键作用。促甲状腺激素释放激素（pro-TRH）前体（26 kDa）在两个位点之一进行蛋白水解切割，产生15/10-kDa或9.5/16.5-kDa的N/C端中间产物对。利用转染了编码前促甲状腺激素释放激素cDNA的AtT20细胞，我们先前报道这组初始切割发生在进入分泌颗粒之前（尼尔尼，E. A.，塞瓦里诺，K. A.，和杰克逊，I. M. D.（1993年）《内分泌学》132，1271 - 1277）。在本研究中，我们着手确定这种初始切割发生的亚细胞区室，并确定所产生中间产物的加工位点。我们的策略是阻断pro-TRH或其中间产物从一个亚细胞区室到下一个区室的转运，并检测中间产物的积累，推测是因为它们的加工发生在阻断后的区室中。在存在布雷菲德菌素A（一种阻断从内质网到高尔基体复合体转运的药物）的情况下，对AtT20细胞进行放射性标记实验，导致26-kDa前体积累，表明加工位点在内质网之后。当在20℃阻断高尔基体复合体到分泌颗粒的转运时，26-kDa前体的加工不受影响，表明加工位点在高尔基体复合体。在此温度下，15-kDa的N端中间产物积累，表明加工位点在高尔基体复合体之后，而16.5-kDa的C端中间产物在高尔基体复合体中加工产生一个5.4-kDa的肽。
