Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2023 |
| Outros Autores: | , , , , |
| Tipo de documento: | Artigo |
| Idioma: | eng |
| Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
| Texto Completo: | https://hdl.handle.net/1822/84430 |
Resumo: | Supplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2023.04.025. |
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Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196Carboxylic acidsExportHeterologous expression in yeastImportSite-directed mutagenesisCiências Naturais::Ciências BiológicasSupplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2023.04.025.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.This work was supported by the Strategic Programme UID/BIA/04050/2020 and the project LA/P/0069/2020 granted to the Associate Laboratory ARNET, both funded by Portuguese funds through the FCT-IP. J.A. acknowledges the FCT and the Doctoral Program in Applied and Environmental Microbiology for the PD/BD/150584/2020 PhD grant and a COST Action CA18113 Short-Term Scientific Mission grant (EuroMicropH). M.S.S. acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the re ference NORTE-08-5369-FSE-000060. I.S-S. was supported by the program contract FCTUMINHO/Norma transitória from the Legal Regime of Scientific Employment (RJEC).ElsevierUniversidade do MinhoAlves, J.Sousa-Silva, M.Soares, P.Sauer, M.Casal, MargaridaSoares-Silva, Isabel João2023-04-262023-04-26T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/1822/84430engAlves, 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.2023.04.0252001-037010.1016/j.csbj.2023.04.025https://www.sciencedirect.com/science/article/pii/S2001037023001782info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:02:17Zoai:repositorium.sdum.uminho.pt:1822/84430Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T18:52:14.836554Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
| dc.title.none.fl_str_mv |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 |
| title |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 |
| spellingShingle |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 Alves, J. Carboxylic acids Export Heterologous expression in yeast Import Site-directed mutagenesis Ciências Naturais::Ciências Biológicas |
| title_short |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 |
| title_full |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 |
| title_fullStr |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 |
| title_full_unstemmed |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 |
| title_sort |
Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196 |
| author |
Alves, J. |
| author_facet |
Alves, J. Sousa-Silva, M. Soares, P. Sauer, M. Casal, Margarida Soares-Silva, Isabel João |
| author_role |
author |
| author2 |
Sousa-Silva, M. Soares, P. Sauer, M. Casal, Margarida Soares-Silva, Isabel João |
| author2_role |
author author author author author |
| dc.contributor.none.fl_str_mv |
Universidade do Minho |
| dc.contributor.author.fl_str_mv |
Alves, J. Sousa-Silva, M. Soares, P. Sauer, M. Casal, Margarida Soares-Silva, Isabel João |
| dc.subject.por.fl_str_mv |
Carboxylic acids Export Heterologous expression in yeast Import Site-directed mutagenesis Ciências Naturais::Ciências Biológicas |
| topic |
Carboxylic acids Export Heterologous expression in yeast Import Site-directed mutagenesis Ciências Naturais::Ciências Biológicas |
| description |
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.csbj.2023.04.025. |
| publishDate |
2023 |
| dc.date.none.fl_str_mv |
2023-04-26 2023-04-26T00:00:00Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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https://hdl.handle.net/1822/84430 |
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https://hdl.handle.net/1822/84430 |
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eng |
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eng |
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Alves, 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 2001-0370 10.1016/j.csbj.2023.04.025 https://www.sciencedirect.com/science/article/pii/S2001037023001782 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Elsevier |
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Elsevier |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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