Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.
Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.
复制标题
抑制早期但非晚期蛋白水解加工事件会导致盘基网柄菌中溶酶体酶前体形式的错误分选和过度分泌。
DOI：
10.1083/jcb.107.6.2097
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Cardelli,JA
中科院分区：
文献类型：
--
作者：
Richardson,JM;Woychik,NA;Ebert,DL;Dimond,RL;Cardelli,JA
溶酶体酶最初合成为前体多肽，其被蛋白水解切割以产生酶活性蛋白的成熟形式。鉴定参与这一过程的蛋白酶及其细胞内位置将是确定蛋白水解在溶酶体酶的功能和靶向中的作用的重要初始步骤。为此，无菌生长的盘基网柄藻细胞脉冲放射性标记与[35 S]蛋氨酸和追逐新鲜的生长培养基含有天冬氨酸，金属，丝氨酸，或半胱氨酸蛋白酶的抑制剂。暴露于丝氨酸/半胱氨酸蛋白酶抑制剂亮抑酶肽和抗痛剂以及半胱氨酸蛋白酶抑制剂苄氧羰基-L-苯丙氨酰-L-丙氨酸-重氮甲基酮（Z-Phe-AlaCHN 2）的细胞无法完成新合成的溶酶体酶、α-甘露糖苷酶和β-葡糖苷酶的蛋白水解加工。抗疼痛药和亮抑酶肽治疗导致蛋白水解加工效率显著降低，以及α-甘露糖苷酶和β-葡糖苷酶前体分泌增加7倍。然而，亮抑酶肽和antipain没有刺激分泌的溶酶体本地化的成熟形式的酶，这表明这些抑制剂防止正常的溶酶体酶前体的溶酶体分选。与用亮抑酶肽或抗痛剂处理的细胞观察到的结果相反，Z-Phe-AlaCHN 2没有阻止前体多肽裂解为酶的中间形式，但大大抑制了成熟酶的产生。然而，积累的中间形式的酶，定位于溶酶体。最后，在Percoll梯度上的细胞提取物的分级表明，α-甘露糖苷酶和β-葡糖苷酶的放射性标记的前体形式到中间产物的加工开始于高尔基复合体和成熟溶酶体之间的密度中间的细胞隔室。相反，成熟形式的生成在到达溶酶体后立即或不久完成。总之，这些结果表明，不同的蛋白酶驻留在不同的细胞内隔室可能参与产生中间和成熟形式的溶酶体酶在盘基网柄藻，和初始裂解的前体可能是至关重要的适当的本地化的溶酶体酶。
Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.