Utilize este identificador para referenciar este registo: https://hdl.handle.net/1822/84430

TítuloStructural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196
Autor(es)Alves, J.
Sousa-Silva, M.
Soares, P.
Sauer, M.
Casal, Margarida
Soares-Silva, Isabel João
Palavras-chaveCarboxylic acids
Export
Heterologous expression in yeast
Import
Site-directed mutagenesis
Data26-Abr-2023
EditoraElsevier
RevistaComputational and Structural Biotechnology Journal
CitaçãoAlves, J., Sousa-Silva, M., Soares, P., Sauer, M., Casal, M., & Soares-Silva, I. (2023). Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196. Computational and Structural Biotechnology Journal. Elsevier BV. http://doi.org/10.1016/j.csbj.2023.04.025
Resumo(s)The Aspergillus niger 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 Saccharomyces cerevisiae, 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. S. cerevisiae 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 Yarrowia lipolytica cex1Δ 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.
TipoArtigo
DescriçãoSupplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2023.04.025.
URIhttps://hdl.handle.net/1822/84430
DOI10.1016/j.csbj.2023.04.025
e-ISSN2001-0370
Versão da editorahttps://www.sciencedirect.com/science/article/pii/S2001037023001782
Arbitragem científicayes
AcessoAcesso aberto
Aparece nas coleções:CBMA - Artigos/Papers

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