title: Functional characterization of two novel purine transporters from the Basidiomycota Phanerochaete chrysosporium.
abstract: Purine transporters as substrate entry points in organisms, are involved in a number of cellular processes such as nitrogen source uptake, energy metabolism and synthesis of nucleic acids. In this study, two nucleobase transporter genes (phZ, phU) from Phanerochaete chrysosporium were cloned, identified, and functionally characterized. Our results show that PhZ is a transporter of adenine and hypoxanthine, and a protein belonging to the AzgA-like family, whilst PhU belongs to the NAT/NCS2 family, transporting xanthine and uric acid. No other sequences belonging to these families were detected in P. chrysosporium's genome. Phylogenetic analyses show that AzgA-like sequences form monophyletic groups for each major lineage (Ascomycota, Basidiomycota and Zygomycota). In contrast, Ascomycota and Basidiomycota NAT/NCS2 sequences do not form monophyletic groups and several copies of this protein are distributed across the tree. Expression of phU was significantly downregulated in the presence of a primary source like ammonium, and enhanced if purines were present or if the mycelium was nitrogen starved. phZ was clearly induced by its substrates (hypoxanthine, adenine), very lightly induced by xanthine, suppressed by urea and amino acids and expressed at a basal level when uric acid or ammonium was the nitrogen source or when the mycelium was starved for nitrogen. In order to perform substrate analyses, both P. chrysosporium proteins (PhZ, PhU) were expressed in Aspergillus nidulans. Epifluorescent microscopy showed that under inducing conditions, PhZ-GFP and PhU-GFP were present at the plasma membrane of A. nidulans transformed strains, and were internalized in repressed conditions. Our results suggest that in the white-rot fungus P. chrysosporium, phU has a catabolic role and phZ, (less dependent of the nitrogen source), plays a key role in purine acquisition to provide biosynthetic components. These are the first purine transporters characterized in Basidiomycota.
authors.affiliation: Microbiología Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay. Electronic address: mariveba@gmail.com.
authors.forename: Mariana
authors.initials: M
authors.lastname: Barraco-Vega
authors.affiliation: Laboratorio de Organización y Evolución del Genoma, Departamento de Ecología y Evolución, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay.
authors.forename: Héctor
authors.initials: H
authors.lastname: Romero
authors.affiliation: Microbiología Instituto de Química Biológica, Facultad de Ciencias - Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay.
authors.forename: Mariana
authors.initials: M
authors.lastname: Richero
authors.affiliation: Microbiología Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay.
authors.forename: María Pía
authors.initials: MP
authors.lastname: Cerdeiras
authors.affiliation: Microbiología Instituto de Química Biológica, Facultad de Ciencias - Facultad de Química, Universidad de la República, Montevideo 11800, Uruguay.
authors.forename: Gianna
authors.initials: G
authors.lastname: Cecchetto
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D020075:Phanerochaete / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011687:Purines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011687:Purines; D011993:Recombinant Fusion Proteins
keywords: Catabolism; Heterologous expression; Nitrogen regulation