Uchida K, Mori H, Mizushima S
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
J Biol Chem. 1995 Dec 29;270(52):30862-8. doi: 10.1074/jbc.270.52.30862.
Derivatives of proOmpA possessing the second cysteine residue at position +302 and the first one at different positions were constructed at the DNA level. They were oxidized to form disulfide-bridged loops of different sizes at different positions. In the presence of a protonmotive force, proOmpAs possessing a smaller loop could be translocated across the membrane in vitro, whereas ones possessing loops comprising more than 16 amino acid residues were hard to translocate. The sizes of polypeptide chains that had been translocated and had become protease-resistant were determined in both the presence and absence of the protonmotive force. The size was the same for all proOmpAs possessing the first cysteine residue between +244 (proOmpA L59) and +274 (proOmpA L29). When the first cysteine residue was moved further away from the N terminus, a sudden increase in size, of approximately 30 amino acid residues, was observed, the size being the same for proOmpAs possessing the first cysteine residue between +278 (proOmpA L25) and +293 (proOmpA L10). The shift in size between proOmpA L29 and proOmpA L25 was observed with different proteases exhibiting different substrate specificities. Treatment with these proteases resulted in complete digestion of SecA on everted membrane vesicles, whereas Sec proteins integrated into membranes were considerably resistant to the treatment. These results can be best interpreted as that the translocation of preproteins through the secretory machinery takes place in every 30 amino acid residues and that SecA is responsible for the stepwise movement.
在DNA水平构建了proOmpA的衍生物，这些衍生物在+302位具有第二个半胱氨酸残基，且第一个半胱氨酸残基位于不同位置。它们被氧化以在不同位置形成不同大小的二硫键桥环。在质子动力势存在的情况下，具有较小环的proOmpA可以在体外跨膜转运，而具有包含超过16个氨基酸残基的环的proOmpA则难以转运。在有和没有质子动力势的情况下，都测定了已转运且具有蛋白酶抗性的多肽链的大小。对于所有在+244（proOmpA L59）和+274（proOmpA L29）之间具有第一个半胱氨酸残基的proOmpA，其大小是相同的。当第一个半胱氨酸残基进一步远离N端时，观察到大小突然增加约30个氨基酸残基，对于在+278（proOmpA L25）和+293（proOmpA L10）之间具有第一个半胱氨酸残基的proOmpA，其大小是相同的。在具有不同底物特异性的不同蛋白酶作用下，观察到了proOmpA L29和proOmpA L25之间的大小变化。用这些蛋白酶处理导致外翻膜囊泡上的SecA完全消化，而整合到膜中的Sec蛋白对该处理具有相当的抗性。这些结果可以最好地解释为前体蛋白通过分泌机制的转运每30个氨基酸残基进行一次，并且SecA负责逐步移动。
