Gutsche J, Remminghorst U, Rehm B H A
Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Biochimie. 2006 Mar-Apr;88(3-4):245-51. doi: 10.1016/j.biochi.2005.06.003. Epub 2005 Jun 23.
AlgX was found to be an essential protein for alginate biosynthesis, but its function is unknown. In this study, an isogenic, marker-free algX-knock out mutant was generated. In-frame fusions of algX with phoA and lacZ were analysed, respectively. No LacZ-activity was detected, but the PhoA fusion showed alkaline phosphatase activity. These data indicated that the C-terminus of AlgX is located in the periplasm, but is not required for protein function. Accordingly, AlgX with C-terminal fusion of strep tag II restored alginate production in the algX-negative mutant and was purified under native conditions from periplasmic and crude cell extracts, respectively. AlgX was identified by MALDI/TOF-MS analysis of tryptic peptides. TritonX-100 mediated solubilisation of cytoplasmic membrane and subsequent strep tag II affinity chromatography led to purification of an AlgX-MucD (AlgY) protein complex as identified by MALDI/TOF-MS analysis. This data suggested a protein-protein interaction between AlgX and MucD (AlgY) with a 1:1 stoichiometry. Thus AlgX might exert its function via interaction with MucD (AlgY). Immunoelectron microscopic localisation of AlgX-strep tag II suggested a localisation close to the cytoplasmic membrane.
AlgX被发现是藻酸盐生物合成所必需的蛋白质，但其功能尚不清楚。在本研究中，构建了一个无标记的同源algX基因敲除突变体。分别分析了algX与phoA和lacZ的读码框内融合情况。未检测到LacZ活性，但PhoA融合蛋白显示出碱性磷酸酶活性。这些数据表明，AlgX的C末端位于周质中，但对蛋白质功能并非必需。因此，C末端融合有链霉亲和标签II的AlgX在algX阴性突变体中恢复了藻酸盐的产生，并分别在天然条件下从周质提取物和粗细胞提取物中纯化出来。通过对胰蛋白酶肽段的MALDI/TOF-MS分析鉴定了AlgX。TritonX-100介导的细胞质膜溶解以及随后的链霉亲和标签II亲和层析导致纯化出一种AlgX-MucD（AlgY）蛋白复合物，通过MALDI/TOF-MS分析得以鉴定。这些数据表明AlgX与MucD（AlgY）之间存在1:1化学计量比的蛋白质-蛋白质相互作用。因此，AlgX可能通过与MucD（AlgY）相互作用发挥其功能。AlgX-链霉亲和标签II的免疫电子显微镜定位表明其定位靠近细胞质膜。
