Smith W P
J Bacteriol. 1980 Mar;141(3):1142-7. doi: 10.1128/jb.141.3.1142-1147.1980.
Crude messenger ribonucleic acid fractions isolated from Corynebacterium diphtheriae and Escherichia coli were translated in an E. coli in vitro protein-synthesizing system and yielded precursors of the secreted proteins diphtheria toxin and alkaline phosphatase, respectively. Addition of inverted E. coli inner membrane vesicles to the system during the initial stages of translation resulted in the intravesicular segregation of mature diphtheria toxin and alkaline phosphatase. Outer membrane vesicles or inner membrane vesicles whose cytoplasmic surfaces had been treated with pronase could not mediate transmembrane transfer of diphtheria toxin or alkaline phosphatase. However, inner membrane vesicles isolated from E. coli spheroplasts which had been treated with pronase and inner membrane vesicles complexed with ribosomes during pronase treatment were functional in transmembrane transfer. At temperatures below the phase transition of E. coli membranes, no intravesicular segregation of alkaline phosphatase or diphtheria toxin was observed. The precursor forms of each protein accumulated free from the vesicles. These results suggest that an inner membrane protein, exposed on the cytoplasmic surface, plays an integral role in secretion.
从白喉棒状杆菌和大肠杆菌中分离得到的粗信使核糖核酸级分，在大肠杆菌体外蛋白质合成系统中进行翻译，分别产生了分泌蛋白白喉毒素和碱性磷酸酶的前体。在翻译初始阶段向系统中添加倒置的大肠杆菌内膜囊泡，导致成熟的白喉毒素和碱性磷酸酶在内囊泡中分离。外膜囊泡或其胞质表面已用链霉蛋白酶处理的内膜囊泡不能介导白喉毒素或碱性磷酸酶的跨膜转运。然而，从经链霉蛋白酶处理的大肠杆菌原生质球中分离得到的内膜囊泡以及在链霉蛋白酶处理期间与核糖体复合的内膜囊泡在跨膜转运中具有功能。在低于大肠杆菌膜相变温度时，未观察到碱性磷酸酶或白喉毒素在内囊泡中的分离。每种蛋白质的前体形式游离于囊泡之外积累。这些结果表明，暴露于胞质表面的一种内膜蛋白在分泌过程中起着不可或缺的作用。
