Ohno-Iwashita Y, Wickner W
J Biol Chem. 1983 Feb 10;258(3):1895-900.
The leader peptidase of Escherichia coli cleaves a 23-residue leader sequence from M13 procoat to yield mature coat protein in virus-infected cells. We have reconstituted pure leader peptidase into vesicles of E. coli lipids and found that these liposomes are active in the conversion of procoat to coat. Trypsin removes all but 10% of the leader peptidase, yet the vesicles retain nearly full capacity to convert procoat to coat, suggesting that only procoat which inserts across the liposomal membrane is a substrate for leader peptidase. This is confirmed by the finding that over 70% of the coat protein produced by these liposomes spans the membrane. The rate at which leader peptidase inside protease-treated liposomes cleaves externally added procoat is comparable to the rate of procoat cleavage by the same amount of leader peptidase in detergent micelles. Thus, procoat can rapidly integrate across a liposomal membrane and be cleaved to coat protein. These findings confirm the central part of the membrane trigger hypothesis that certain proteins (such as procoat) can cross a bilayer without the aid of a proteinaceous pore or transport system.
大肠杆菌的前导肽酶在病毒感染的细胞中，从M13原衣壳蛋白上切割下一段23个残基的前导序列，产生成熟的衣壳蛋白。我们已将纯化的前导肽酶重组到大肠杆菌脂质囊泡中，发现这些脂质体能够将原衣壳蛋白转化为衣壳蛋白。用胰蛋白酶处理后，除了10%的前导肽酶外，其余的都被去除，但脂质体仍保留了将近全部将原衣壳蛋白转化为衣壳蛋白的能力，这表明只有跨脂质体膜插入的原衣壳蛋白才是前导肽酶的底物。超过70%由这些脂质体产生的衣壳蛋白跨膜存在，这一发现证实了上述观点。经蛋白酶处理的脂质体内的前导肽酶切割外部添加的原衣壳蛋白的速率，与相同量的前导肽酶在去污剂胶束中切割原衣壳蛋白的速率相当。因此，原衣壳蛋白能够快速整合穿过脂质体膜并被切割成衣壳蛋白。这些发现证实了膜触发假说的核心内容，即某些蛋白质（如原衣壳蛋白）可以在没有蛋白质孔道或转运系统帮助的情况下穿过双层膜。
