Burgess T L, Craik C S, Matsuuchi L, Kelly R B
J Cell Biol. 1987 Aug;105(2):659-68. doi: 10.1083/jcb.105.2.659.
The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.
小鼠垂体前叶肿瘤细胞系AtT-20将分泌蛋白靶向两种不同的细胞内途径。当将编码胰蛋白酶原的DNA导入AtT-20细胞时，该蛋白与内源性肽激素促肾上腺皮质激素（ACTH）一样有效地被分选到调节性分泌途径中。在本研究中，我们使用双标记免疫电子显微镜来证明胰蛋白酶原与ACTH共定位于相同的分泌颗粒中。体外诱变用于测试将胰蛋白酶原靶向分泌颗粒的信息是否位于该蛋白的氨基（NH2）末端。对编码胰蛋白酶原的DNA进行突变，并在AtT-20细胞中表达突变蛋白，以确定细胞内靶向是否可以改变。用组成性分泌蛋白κ-免疫球蛋白轻链的信号肽取代胰蛋白酶原信号肽，不会改变对调节性分泌途径的靶向。此外，删除胰蛋白酶原的NH2末端“前体”序列对蛋白质靶向几乎没有影响。然而，这种删除确实影响信号肽酶切割位点，结果截短的胰蛋白酶蛋白的酶活性被消除。我们得出结论，胰蛋白酶原的信号肽和12个NH2末端氨基酸对于分选到AtT-20细胞的调节性分泌途径都不是必需的。
