Study of molecular mechanism of protein translocation through biochemical analysis of mutant secY in Escherichia coli

在大肠杆菌蛋白质易位分析领域，对多种sec突变体进行体外分析，特别是在纯化形式下比较它们的生化性质，是最迫切的主题。本课题的目的是纯化野生型和突变型SecY蛋白单体以及与SecE和SecG形成的复合物，利用His_6-taging方法成功地纯化了野生型SecY和6种突变型SecY，包括SecY的cs等位基因SecY 39，SecYEG复合物和野生型SecE单体。通过该方法纯化的野生型SecYEG复合物保留了通过重建SecYEG蛋白脂质体测量的蛋白质易位活性。下一步，我将从以下几个方面对纯化后的配合物进行分析和比较; 1）对分泌前体的亲和力，2）对SecA ATP酶的亲和力，3）对SecA的膜插入和去插入的影响，和4）通过分析Ni^<2+>抑制His_6-taged pro-1的作用，研究Ni^<2+>对蛋白质转运早期阶段的影响。OmpA易位：在我使用的分泌前体的模型蛋白--带His_6标签的pro-OmpA的纯化和蛋白易位分析过程中，我发现如果将His_6标签插入成熟OmpA结构域的N端区域，则其易位获得Ni^<2+>敏感性。Ni^<2+>的抑制作用被组氨酸中和，并通过使用由prlA 3制备的膜囊泡来中和，prlA 3是secY的等位基因，被认为拓宽了移位酶的信号识别特异性。Ni^（2+）仅在蛋白质转位的后靶向阶段有效，并且还抑制了pro-OmpA和SecY之间的化学交联，这些结果表明Sec YEG复合物在转位的早期直接识别分泌前体的可能性。

In the field of protein translocation analyzes in Escherichia coli, it is the most urgent theme to analyze a variety of sec mutants in vitro, especially, to compare their biochemical nature in a purified form. My aim in this project is purification of wild type and mutant SecY proteins both in a monomer and a complex with SecE and SecG.Using His_6-tagging method, I succeeded to purify wild type and 6 mutant forms of SecY including SecY39, a cs allele of secY,in SecYEG complexes and the wild type SecE monomer. The purified wild type SecYEG complex by this procedure retained protein translocation activity measured by reconstitution of SecYEG proteoliposome. Now I am trying, as the next step, to analyze and compare the purified complexes from the point of the following views ; 1) affinity to secretory precursors, 2) affinity to SecA ATPase, 3) effects on the membrane insertion and de-insertion of SecA,and 4) effects on an early stage of protein translocation through the analysis of Ni^<2+> inhibition of His_6-tagged pro-OmpA translocation.During the purification and protein translocation analysis of His_6-tagged pro-OmpA,a model protein of secretory precursor I used, I found that its translocation acquired Ni^<2+> sensitivity if a His_6 tag is inserted into the N-terminal region of the mature OmpA domain. The inhibitory effect by Ni^<2+> is neutralized by histidine and by using membrane vesicles prepared from prlA3, an allele of secY that is thought to broaden signal recognition specificity of translocase. Ni^<2+> is only effective in a post-targeting stage of the protein translocation, and also inhibited chemical cross-linking between pro-OmpA and SecY.These results demonstrated a possibility that Sec YEG complex directly recognizes secretory precursors in an early stage of the translocation.