Yost C S, Hedgpeth J, Lingappa V R
Cell. 1983 Oct;34(3):759-66. doi: 10.1016/0092-8674(83)90532-9.
We have combined molecular genetic and cell-free reconstitution approaches to study the mechanism of membrane assembly. The coding region for the carboxy-terminal transmembrane sequence of membrane IgM heavy chain has been inserted between the coding regions for lactamase and globin domains of a fusion protein previously shown to be completely translocated across microsomal membranes in a cell-free transcription-linked translation system. The resulting fusion protein behaves as an integral transmembrane protein of predicted asymmetry: all of the membrane integrated copies display lactamase within the lumen and globin on the cytoplasmic face of the vesicles. In another construction, this transmembrane coding region replaces that of the signal sequence. The resulting fusion protein is not translocated across membranes. These data provide strong evidence that there are stop transfer sequences whose ability to arrest chain translocation and achieve asymmetric transmembrane orientation is independent of the size of the subsequent carboxy-terminal domain to be localized in the cytosol; and that signal and stop transfer sequences are functionally distinct.
我们结合了分子遗传学和无细胞重建方法来研究膜组装机制。膜IgM重链羧基末端跨膜序列的编码区已插入到一种融合蛋白的内酰胺酶和球蛋白结构域的编码区之间，该融合蛋白先前已在无细胞转录偶联翻译系统中被证明能完全跨微粒体膜转运。所得融合蛋白表现为具有预测不对称性的整合跨膜蛋白：所有整合到膜中的拷贝在内腔中显示内酰胺酶，在囊泡的细胞质面上显示球蛋白。在另一种构建中，该跨膜编码区取代了信号序列的编码区。所得融合蛋白不能跨膜转运。这些数据提供了强有力的证据，即存在终止转移序列，其阻止链转运并实现不对称跨膜取向的能力与随后定位于胞质溶胶中的羧基末端结构域的大小无关；并且信号序列和终止转移序列在功能上是不同的。
