Richardson J M, Woychik N A, Ebert D L, Dimond R L, Cardelli J A
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130.
J Cell Biol. 1988 Dec;107(6 Pt 1):2097-107. doi: 10.1083/jcb.107.6.2097.
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.
溶酶体酶最初作为前体多肽合成，这些前体多肽经蛋白水解切割后产生具有酶活性的成熟蛋白形式。鉴定参与这一过程的蛋白酶及其细胞内定位，将是确定蛋白水解在溶酶体酶功能和靶向中的作用的重要初始步骤。为此，用[35S]甲硫氨酸对无菌培养的盘基网柄菌细胞进行脉冲放射性标记，并在含有天冬氨酸、金属、丝氨酸或半胱氨酸蛋白酶抑制剂的新鲜生长培养基中进行追踪。暴露于丝氨酸/半胱氨酸蛋白酶抑制剂亮抑酶肽和抗蛋白酶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-AlaCHN2）的细胞，无法完成新合成的溶酶体酶α-甘露糖苷酶和β-葡萄糖苷酶的蛋白水解加工。抗蛋白酶和亮抑酶肽处理导致蛋白水解加工效率急剧下降，同时α-甘露糖苷酶和β-葡萄糖苷酶前体的分泌增加了七倍。然而，亮抑酶肽和抗蛋白酶并未刺激溶酶体定位的酶成熟形式的分泌，这表明这些抑制剂阻止了溶酶体酶前体正常分选到溶酶体中。与用亮抑酶肽或抗蛋白酶处理的细胞所观察到的结果相反，Z-Phe-AlaCHN2并未阻止前体多肽切割成酶的中间形式，但极大地抑制了成熟酶的产生。然而，积累的酶中间形式定位于溶酶体中。最后，在Percoll梯度上对细胞提取物进行分级分离表明，放射性标记的α-甘露糖苷酶和β-葡萄糖苷酶前体形式加工成中间产物始于高尔基体复合体和成熟溶酶体之间密度中等的细胞区室。相比之下，成熟形式的产生在到达溶酶体时或之后立即完成。这些结果共同表明，存在于不同细胞内区室的不同蛋白酶可能参与了盘基网柄菌中溶酶体酶中间形式和成熟形式的产生，并且前体的初始切割可能对溶酶体酶的正确定位至关重要。
