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

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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.