Milgram S L, Eipper B A, Mains R E
Neuroscience Department, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Cell Biol. 1994 Jan;124(1-2):33-41. doi: 10.1083/jcb.124.1.33.
The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.
翻译后加工酶肽基甘氨酸α-酰胺化单加氧酶（PAM）以整合膜形式和可溶性形式天然存在。为了了解这些蛋白质靶向分泌颗粒的机制，我们比较了稳定转染的AtT-20细胞中PAM蛋白的成熟、加工、分泌和储存情况。整合膜PAM蛋白和可溶性PAM蛋白以相似的动力学离开内质网并到达高尔基体。生物合成标记实验表明，可溶性PAM蛋白比整合膜PAM蛋白在内切蛋白水解加工方面程度更高；这种加工发生在调节性分泌途径中，并在20℃孵育细胞时被阻断。生物合成脉冲16小时后，与整合膜PAM相比，可溶性PAM蛋白与细胞相关的比例更大，这表明可溶性PAM蛋白更有效地靶向储存颗粒。可溶性PAM蛋白的非刺激分泌在生物合成脉冲后1-2小时达到峰值，表明释放来自在成熟过程中从不成熟颗粒出芽的小泡。相反，通过整合膜PAM的内切蛋白水解裂解产生的可溶性PAM蛋白在追踪后期分泌量最高。此外，细胞表面相关整合膜PAM的免疫沉淀表明，在追踪早期很少有整合膜PAM到达细胞表面。然而，当在AtT-20细胞中表达缺乏细胞质尾的截短PAM蛋白时，>50%的截短PAM-1蛋白在3小时内到达细胞表面。我们得出结论，整合膜和可溶性分泌颗粒相关酶的运输不同，并且整合膜PAM蛋白在成熟分泌颗粒中的保留效率较低。
