The proregion of cathepsin L is required for proper folding, stability, and ER exit.
The proregion of cathepsin L is required for proper folding, stability, and ER exit.
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DOI：
10.1006/abbi.1994.1203
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发表时间：
1994-05
影响因子：
3.9
通讯作者：
K. Tao;N. A. Stearns;J. Dong;Q. L. Wu;G. Sahagian
中科院分区：
文献类型：
--
作者：
K. Tao;N. A. Stearns;J. Dong;Q. L. Wu;G. Sahagian
为了研究前区在小鼠组织蛋白酶L的生物合成和运输中的作用，构建了编码具有改变的前区的蛋白质的组织蛋白酶L cDNA，并检测了它们在COS细胞中的表达。与转化细胞一样，COS细胞分泌正常小鼠组织蛋白酶L。相比之下，两种改变的前区组织蛋白酶L蛋白，其中一种前区缺失，第二种前区被同源蛋白（糊粉蛋白）的前区取代，保留在细胞内并在2-6小时内降解。免疫荧光定位和缺乏NH 4Cl和布雷菲德菌素A对改变的组织蛋白酶L蛋白的营业额的影响表明，它们的降解发生在内质网（ER）。通过使用布雷菲德菌素A诱导UDPGlcNAc：溶酶体酶N-乙酰葡糖胺-1-磷酸转移酶与ER中的组织蛋白酶L蛋白的共定位，表明改变的蛋白质不受甘露糖磷酸化的影响，因为它们存在于ER中。胰蛋白酶敏感性试验表明，在COS细胞或体外合成的改变的前区蛋白质是错误折叠的。综上所述，这些结果表明前区在组织蛋白酶L的正确折叠中起重要作用。ER滞留、稳定性降低和改变的蛋白质缺乏甘露糖磷酸化最有可能是由不正确折叠引起的继发性效应。
To investigate the role of the proregion in the biosynthesis and trafficking of mouse cathepsin L, cathepsin L cDNAs encoding proteins with altered proregions were constructed and their expression in COS cells was examined. As in transformed cells, normal mouse cathepsin L was secreted by COS cells. In contrast, two altered proregion cathepsin L proteins, one in which the proregion was deleted and a second in which the proregion was replaced with that of a homologous protein (aleurain), were retained within the cell and degraded over a period of 2-6 h. Immunofluorescence localization and the lack of effect of NH4Cl and brefeldin A on the turnover of the altered cathepsin L proteins indicated that their degradation occurred in the endoplasmic reticulum (ER). By using brefeldin A to induce colocalization of the UDPGlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase with the cathepsin L proteins in the ER, it was shown that the altered proteins were not susceptible to mannose phosphorylation as they exist in the ER. Trypsin sensitivity assays indicated that altered proregion proteins synthesized in COS cells or in vitro are misfolded. Taken together, these results indicate that the proregion plays an essential role in proper folding of cathepsin L. ER retention, decreased stability, and lack of mannose phosphorylation of the altered proteins are most likely secondary effects resulting from improper folding.