Alves J, Sousa-Silva M, Soares P, Sauer M, Casal M, Soares-Silva I
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Comput Struct Biotechnol J. 2023 Apr 26;21:2884-2898. doi: 10.1016/j.csbj.2023.04.025. eCollection 2023.
The CexA transporter belongs to the DHA1 (Drug-H antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in , demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the 1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity.
CexA转运蛋白属于DHA1（药物-H反向转运蛋白）家族。CexA同源物仅在真核生物基因组中发现，并且CexA是迄今为止在该家族中唯一已被功能表征的柠檬酸盐输出蛋白。在本研究中，我们在[具体物种]中表达了CexA，证明了其结合异柠檬酸的能力，并在pH 5.5时以低亲和力导入柠檬酸盐。柠檬酸盐的摄取与质子动力势无关，并且与促进扩散机制一致。为了阐明该转运蛋白的结构特征，我们随后对21个CexA残基进行了定点诱变。通过DHA1家族中氨基酸残基保守性、三维结构预测和底物分子对接分析相结合的方法确定了这些残基。对表达该CexA突变等位基因文库的[具体物种]细胞在含羧酸培养基上生长的能力以及放射性标记柠檬酸盐的转运进行了评估。我们还通过绿色荧光蛋白（GFP）标记确定了蛋白质的亚细胞定位，有七个氨基酸取代影响了CexA蛋白在质膜上的表达。P200A、Y307A、S315A和R461A取代表现出功能丧失的表型。大多数取代影响了柠檬酸盐的结合和转运。S75残基对柠檬酸盐输出没有影响，但影响其输入，因为用丙氨酸取代增加了转运蛋白对柠檬酸盐的亲和力。相反，在[具体物种]1Δ菌株中表达CexA突变等位基因揭示了R192和Q196残基参与柠檬酸盐输出。总体而言，我们发现了一组与CexA表达、输出能力和输入亲和力相关的氨基酸残基。
