Título:
Mutational analysis of Phanerochaete chrysosporium's purine transporter
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Autor:
Barraco Vega, Mariana
Sanguinetti, Manuel
da Rosa, Gabriela
Cecchetto, Gianna
Sanguinetti, Manuel
da Rosa, Gabriela
Cecchetto, Gianna
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MEMBRANAS CELULARES
PURINAS
FENOTIPOS
ASPERGILLUS NIDULANS
PURINAS
FENOTIPOS
ASPERGILLUS NIDULANS
Año de publicación:
2024
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Resumen:
We present here a mutational analysis of the purine transporter from Phanerochaete chrysosporium (PhZ), a member of the AzgA-like subfamily within the Nucleobase Ascorbate Transporters family. We identified key residues that determine its substrate specificity and transport efficiency. Thirteen PhZ mutants were generated and heterologously expressed in Aspergillus nidulans. The growth of mutant strains in the presence of purines and toxic analogues and the uptake rate of radiolabelled hypoxanthine were evaluated. Results revealed that ten mutants showed differences in transport compared to the wild-type PhZ: six mutants completely lost function, two exhibited decreased transport activity, and two showed increased hypoxanthine uptake. Subcellular localization and expression level analyses indicated that the differences in transport activity were not due to trafficking issues to the plasma membrane or protein stability. A three-dimensional model of PhZ, constructed with the artificial intelligence-based AlphaFold2 program, suggested that critical residues for transport are located in transmembrane segments and an internal helix. In the latter, the A418 residue was identified as playing a pivotal role in transport efficiency despite being far from the putative substrate binding site, as mutant A418V showed an increased initial uptake efficiency for the transporter´s physiological substrates. We also report that residue L124, which lies in the putative substrate binding site, plays a critical role in substrate transport, emerging as an additional determinant in the transport mechanism of this family of transporters. These findings underscore the importance of specific residues in AzgA-like transporters and enhance our understanding of the intricate mechanisms governing substrate specificity and transport efficiency within this family.
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PLOS
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PLoS ONE, v. 19, nº. 10, 2024. -- e0313174
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Citación:
Barraco Vega, M., Sanguinetti, M., da Rosa, G. y otro. "Mutational analysis of Phanerochaete chrysosporium's purine transporter". PLoS ONE [en línea] v. 19, nº 10, 2024. -- e0313174. 25 p.
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https://hdl.handle.net/20.500.12008/47467
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Licencia Creative Commons Atribución (CC - By 4.0)
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| Ficheros | Descripción | Tamaño | Formato | ||
|---|---|---|---|---|---|
| Mutational analysis of Phanerochaete.pdf | — | 4.34 MB | Adobe PDF |