Klauser T, Pohlner J, Meyer T F
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, FRG.
EMBO J. 1990 Jun;9(6):1991-9. doi: 10.1002/j.1460-2075.1990.tb08327.x.
The beta-domain of the Neisseria IgA protease precursor (Iga) provides the essential transport function for the protease across the outer membrane. To investigate the secretion function of the beta-domain (Iga beta), we engineered hybrid proteins between Iga beta and the non-toxic 12 kd cholera toxin B subunit (CtxB) and examined their targeting behaviour in Salmonella typhimurium. We show that CtxB-Iga beta hybrid proteins integrate into the outer membrane, leading to the exposition of the CtxB moiety on the cell surface. Exposed CtxB can be degraded by externally added proteases like trypsin, but can also be specifically cleaved off from membrane-associated Iga beta by purified IgA protease. We further demonstrate that folding of the CtxB moiety at the periplasmic side of the outer membrane interferes with its translocation. Prevention of disulphide-induced folding in periplasmic CtxB renders the protein moiety competent for outer membrane transport. Iga beta may be of general interest as an export vehicle for even larger proteins from Gram-negative bacteria.
淋病奈瑟菌IgA蛋白酶前体（Iga）的β结构域为该蛋白酶提供了跨越外膜的必要转运功能。为了研究β结构域（Igaβ）的分泌功能，我们构建了Igaβ与无毒的12kd霍乱毒素B亚基（CtxB）之间的杂合蛋白，并检测了它们在鼠伤寒沙门氏菌中的靶向行为。我们发现CtxB-Igaβ杂合蛋白整合到外膜中，导致CtxB部分暴露于细胞表面。暴露的CtxB可被外部添加的蛋白酶如胰蛋白酶降解，但也可被纯化的IgA蛋白酶从膜相关的Igaβ上特异性切割下来。我们进一步证明，CtxB部分在外膜周质侧的折叠会干扰其转运。防止周质CtxB中二硫键诱导的折叠可使该蛋白部分能够进行外膜转运。Igaβ作为革兰氏阴性菌中更大蛋白质的输出载体可能具有普遍意义。
