Kato M, Tokuda H, Mizushima S
Institute of Applied Microbiology, University of Tokyo, Japan.
J Biol Chem. 1992 Jan 5;267(1):413-8.
The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied. Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein. The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force. Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins. The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain. All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size. The mature domain of the smallest one was 45 amino acid residues in length. Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA.
研究了分泌蛋白成熟结构域上存在的电荷对其转运到大肠杆菌外翻膜囊泡中的效率和质子动力势依赖性的影响。以proOmpF-Lpp作为起始蛋白，在DNA水平构建了成熟结构域无电荷的模型分泌蛋白。如此构建的无电荷前分泌蛋白比proOmpF-Lpp转运和信号肽加工速度快得多，施加质子动力势可显著提高转运速率。不仅膜电位，而且ΔpH都能有效刺激无电荷蛋白的转运速率。结果表明，分泌转运过程中成熟结构域不一定带电荷，质子动力势对分泌转运的主要需求不是针对成熟结构域带电区域的移动（包括电泳移动）。如此构建的所有proOmpF-Lpp衍生物都以依赖SecA的方式有效地转运到外翻膜囊泡中，而与它们的大小无关。最小的一种成熟结构域长度为45个氨基酸残基。与其他研究者先前提出的观点相反，这些结果表明，在前分泌蛋白跨细胞质膜转运的报道区域或SecA需求方面没有明显界限。
