Milgram S L, Mains R E
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
J Cell Sci. 1994 Mar;107 ( Pt 3):737-45. doi: 10.1242/jcs.107.3.737.
Vesicular transport within the secretory pathway can be arrested by incubating cells at 15 degrees C or 20 degrees C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble peptidylglycine alpha-amidating monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4 degrees C, 15 degrees C, 20 degrees C or 37 degrees C. The endoproteolytic processing of peptidylglycine alpha-amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble peptidylglycine alpha-amidating monooxygenase proteins was completely blocked by incubation of cells at 20 degrees C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15 degrees C and 20 degrees C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20 degrees C. In AtT-20 cells and neurointermediate pituitary cultures, generation of beta-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20 degrees C, while processing of beta-lipotropin to beta-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while peptidylglycine alpha-amidating monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.
通过在15℃或20℃孵育细胞，分别阻断从内质网或反式高尔基体网络的输出，可使分泌途径内的囊泡运输停滞。利用这一强大工具，我们比较了阿黑皮素原和两种激素原加工酶在AtT - 20小鼠垂体促肾上腺皮质激素肿瘤细胞内的内切蛋白水解加工位点。作为对照，还在原代神经垂体中间叶培养物中评估了阿黑皮素原的加工情况。AtT - 20细胞合成并储存内源性阿黑皮素原和激素原转化酶1；通过稳定转染产生表达高水平整合膜或可溶性肽基甘氨酸α - 酰胺化单加氧酶的AtT - 20细胞。将细胞与[35S]甲硫氨酸一起孵育，并在4℃、15℃、20℃或37℃进行追踪。免疫沉淀后比较肽基甘氨酸α - 酰胺化单加氧酶、激素原转化酶1和阿黑皮素原的内切蛋白水解加工情况。通过在20℃孵育细胞，整合膜和可溶性肽基甘氨酸α - 酰胺化单加氧酶蛋白的内切蛋白水解加工被完全阻断。相反，激素原转化酶1从87 kDa前体加工为81 kDa中间体在15℃和20℃均能完成，而生成63 kDa激素原转化酶1蛋白的切割在20℃被完全阻断。在AtT - 20细胞和神经垂体中间叶培养物中，在20℃时，阿黑皮素原生成β - 促脂素的过程以缓慢但显著的速率持续进行，而β - 促脂素加工为β - 内啡肽的过程被阻断。因此，激素原转化酶1的加工在内质网中开始，直到反式高尔基体网络之后才完成，而肽基甘氨酸α - 酰胺化单加氧酶的加工在反式高尔基体网络之后开始。选定的阿黑皮素原切割在进入未成熟颗粒之前就开始了。
