The soluble cytoplasmic N-terminal domain of the FocA channel gates bidirectional formate translocation
The soluble cytoplasmic N-terminal domain of the FocA channel gates bidirectional formate translocation
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DOI：
10.1111/mmi.14641
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发表时间：
2020-11-30
影响因子：
3.6
通讯作者：
Sawers, Robert Gary
中科院分区：
文献类型：
--
作者：
Kammel, Michelle;Hunger, Doreen;Sawers, Robert Gary
FocA 属于阴离子通道五聚体 FNT（甲酸-亚硝酸盐转运蛋白）超家族，可将甲酸双向转运穿过大肠杆菌和其他微生物的细胞质膜。虽然 FocA 的膜整合核心与其他 FNT 家族成员具有相当大的氨基酸序列保守性，但可溶性细胞质 N 端结构域却不然。为了分析 FocA N 端结构域在体内的潜在生化功能，我们构建了截短衍生物和氨基酸交换变体，并通过使用甲酸敏感报告系统监测细胞内甲酸水平来确定它们跨大肠杆菌细胞膜转运甲酸的能力。对合成这些 FocA 变体的菌株的分析提供了对甲酸流出的见解。缺乏细胞内产生甲酸能力的菌株使我们能够确定这些变体是否可以输入甲酸或其有毒化学类似物次磷酸盐。我们的研究结果表明，FocA 的 N 端结构域对于依赖 FocA 的双向甲酸跨膜渗透至关重要。此外，我们发现柔性N端结构域的氨基酸序列基序和二级结构特征对于甲酸易位很重要，并且流出/流入受到丙酮酸甲酸裂解酶的影响。因此，可溶性 N 末端结构域对于 FocA 的双向甲酸易位至关重要，表明其具有控制阴离子可及性的“看门人”功能。
FocA belongs to the pentameric FNT (formate-nitrite transporter) superfamily of anion channels, translocating formate bidirectionally across the cytoplasmic membrane of Escherichia coli and other microorganisms. While the membrane-integral core of FocA shares considerable amino acid sequence conservation with other FNT family members, the soluble cytoplasmic N-terminal domain does not. To analyze the potential biochemical function of FocA's N-terminal domain in vivo, we constructed truncation derivatives and amino acid-exchange variants, and determined their ability to translocate formate across the membrane of E. coli cells by monitoring intracellular formate levels using a formate-sensitive reporter system. Analysis of strains synthesizing these FocA variants provided insights into formate efflux. Strains lacking the ability to generate formate intracellularly allowed us to determine whether these variants could import formate or its toxic chemical analog hypophosphite. Our findings reveal that the N-terminal domain of FocA is crucial for bidirectional FocA-dependent permeation of formate across the membrane. Moreover, we show that an amino acid sequence motif and secondary structural features of the flexible N-terminal domain are important for formate translocation, and efflux/influx is influenced by pyruvate formate-lyase. The soluble N-terminal domain is, therefore, essential for bidirectional formate translocation by FocA, suggesting a "gate-keeper" function controlling anion accessibility.