Chang C N, Model P, Blobel G
Proc Natl Acad Sci U S A. 1979 Mar;76(3):1251-5. doi: 10.1073/pnas.76.3.1251.
The coat protein (CP) of bacteriophage f1 is integrated into an Escherichia coli plasma membrane fraction consisting of inverted vesicles when it is synthesized in a cell-free, coupled transcription--translation system supplemented with the inverted vesicles. By using proteolytic enzymes as probes, we found by subsequent peptide mapping and determination of the sequence of the proteolytic products that CP was inserted into the inverted vesicles in an orientation indistinguishable from that in inverted vesicles prepared from infected E. coli: only a COOH-terminal portion of approximately 10 residues was accessible to proteolysis, whereas the remainder of CP (CP') was entirely protected. Protection of CP' was dependent on the integrity of the vesicle membrane, because it was abolished when proteolysis was done in the presence of nonionic detergents. Insertion was observed when the inverted vesicles were present during translation in the cell-free system, not when they were added after translation. Thus, the asymmetric insertion of this type of integral membrane protein is strictly coupled to translation. These findings are discussed with respect to prokaryotic membrane biogenesis and are related to bacteriophage f1 assembly and infection.
当噬菌体f1的外壳蛋白（CP）在添加了反向囊泡的无细胞偶联转录-翻译系统中合成时，它会整合到由反向囊泡组成的大肠杆菌质膜组分中。通过使用蛋白水解酶作为探针，我们通过随后的肽图谱分析和蛋白水解产物序列测定发现，CP以与从感染大肠杆菌制备的反向囊泡中难以区分的方向插入到反向囊泡中：只有大约10个残基的COOH末端部分可被蛋白水解作用所作用，而CP的其余部分（CP'）则完全受到保护。CP'的保护依赖于囊泡膜的完整性，因为在非离子去污剂存在下进行蛋白水解时这种保护作用就会消失。当在无细胞系统翻译过程中存在反向囊泡时可观察到插入现象，而在翻译后添加则不会出现。因此，这种类型的整合膜蛋白的不对称插入与翻译严格偶联。我们就原核生物膜生物发生对这些发现进行了讨论，并将其与噬菌体f1的组装和感染联系起来。
