Simon K, Perara E, Lingappa V R
J Cell Biol. 1987 May;104(5):1165-72. doi: 10.1083/jcb.104.5.1165.
We have studied the translocation of a normally cytoplasmic protein domain across the membrane of the endoplasmic reticulum in cell-free systems and in Xenopus laevis oocytes. Coding regions for the normally cytoplasmic protein globin were engineered in frame either 3' or 5' to the coding region for the signal sequence of either Escherichia coli b-lactamase or bovine preprolactin, respectively, in SP6 expression plasmids. RNA transcribed from these plasmids was microinjected into oocytes as well as translated in cell-free systems. We demonstrate that both in vivo and in vitro, a previously amino-terminal signal sequence can direct translocation of domains engineered to either side. Moreover, the domain preceding the signal sequence can be as large as that which follows it. While, in general, cell-free systems were found to faithfully reflect translocation events in vivo, our results suggest that a mechanism for clearance of signal peptides after cleavage is present in intact cells that is not reconstituted in cell-free systems.
我们已经在无细胞系统和非洲爪蟾卵母细胞中研究了一种正常位于细胞质中的蛋白质结构域穿过内质网膜的转运情况。在SP6表达质粒中，将正常位于细胞质中的球蛋白编码区分别在大肠杆菌β-内酰胺酶或牛催乳素原信号序列编码区的3'端或5'端进行框内构建。从这些质粒转录的RNA被显微注射到卵母细胞中，并在无细胞系统中进行翻译。我们证明，在体内和体外，一个先前位于氨基末端的信号序列都可以指导构建在其两侧的结构域的转运。此外，信号序列之前的结构域可以与之后的结构域一样大。虽然一般来说，无细胞系统被发现能够忠实地反映体内的转运事件，但我们的结果表明，完整细胞中存在一种信号肽切割后清除的机制，而无细胞系统中并未重建该机制。
