Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation
Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation
复制标题
DOI：
10.1016/j.jmb.2014.05.023
复制
发表时间：
2014-07-29
影响因子：
5.6
通讯作者：
Sawers, R. Gary
中科院分区：
文献类型：
--
作者：
Doberenz, Claudia;Zorn, Michael;Sawers, R. Gary
FNT（甲酸盐-亚硝酸盐转运蛋白）形成五聚体膜通道的超家族，其跨生物膜转运单价阴离子。福卡（甲酸通道A）双向转运甲酸，但如何控制甲酸转运的潜在机制以及什么支配底物特异性仍不清楚。在这里，我们表明，通常可溶性二聚体酶丙酮酸甲酸裂解酶（PfIB），这是负责在肠杆菌和其他微生物的细胞内甲酸的产生，特别是与福卡相互作用。PfIB与细胞质膜的结合显示为福卡依赖性的，并且纯化的、Strep标记的福卡特异性地从大肠杆菌粗提取物中回收PfIB。利用细菌双杂交系统，可以表明，福卡的N-末端和PfIB的中心结构域参与相互作用。化学交联实验证实了这一发现。使用的限制所施加的交联研究中确定的氨基酸残基，我们首次提供了一个模型的福卡PfIB复合物。该模型表明，N-末端的福卡是重要的相互作用与PfIB。开发用于监测细胞质中甲酸盐水平变化的体内测定揭示了与PfIB的相互作用对于福卡最佳甲酸盐易位的重要性。该系统代表了FNT通道蛋白活性控制的范例。(C)2014爱思唯尔有限公司版权所有。
The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-Iyase (PfIB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PfIB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PfIB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PfIB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA PfIB complex. The model suggests that the N-terminus of FocA is important for interaction with PfIB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PfIB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins. (C) 2014 Elsevier Ltd. All rights reserved.