Rodrigues Thiago E, Souza Victor E P, Monteiro Rose A, Gerhardt Edileusa C M, Araújo Luíza M, Chubatsu Leda S, Souza Emanuel M, Pedrosa Fábio O, Huergo Luciano F
Department of Biochemistry and Molecular Biology, Universidade Federal do Parana, Curitiba, Brazil.
Biochim Biophys Acta. 2011 Sep;1814(9):1203-9. doi: 10.1016/j.bbapap.2011.05.012. Epub 2011 May 27.
The ammonium transport family Amt/Rh comprises ubiquitous integral membrane proteins that facilitate ammonium movement across biological membranes. Besides their role in transport, Amt proteins also play a role in sensing the levels of ammonium in the environment, a process that depends on complex formation with cytosolic proteins of the P(II) family. Trimeric P(II) proteins from a variety of organisms undergo a cycle of reversible posttranslational modification according to the prevailing nitrogen supply. In proteobacteria, P(II) proteins are subjected to reversible uridylylation of each monomer. In this study we used the purified proteins from Azospirillum brasilense to analyze the effect of P(II) uridylylation on the protein's ability to engage complex formation with AmtB in vitro. Our results show that partially uridylylated P(II) trimers can interact with AmtB in vitro, the implication of this finding in the regulation of nitrogen metabolism is discussed. We also report an improved expression and purification protocol for the A. brasilense AmtB protein that might be applicable to AmtB proteins from other organisms.
铵转运蛋白家族Amt/Rh由普遍存在的整合膜蛋白组成，这些蛋白促进铵跨生物膜的移动。除了在转运中发挥作用外，Amt蛋白还在感知环境中铵的水平方面发挥作用，这一过程依赖于与P(II)家族的胞质蛋白形成复合物。来自多种生物体的三聚体P(II)蛋白根据主要的氮供应经历可逆的翻译后修饰循环。在变形杆菌中，P(II)蛋白的每个单体都会发生可逆的尿苷酸化。在本研究中，我们使用来自巴西固氮螺菌的纯化蛋白来分析P(II)尿苷酸化对该蛋白在体外与AmtB形成复合物能力的影响。我们的结果表明，部分尿苷酸化的P(II)三聚体在体外可与AmtB相互作用，并讨论了这一发现对氮代谢调节的意义。我们还报告了一种改进的巴西固氮螺菌AmtB蛋白表达和纯化方案，该方案可能适用于其他生物体的AmtB蛋白。
