Functional characterization of two novel purine transporters from the Basidiomycota Phanerochaete chrysosporium.

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.

Mutations in the basic loop of the Zn binuclear cluster of the UaY transcriptional activator suppress mutations in the dimerisation domain.

UaY is the specific ZnII(2)Cys(6) transcriptional activator of the purine utilisation pathway in Aspergillus nidulans. Previous work has determined the consensus binding sequence by EMSA and foot-printing. We determine here that it binds as a dimer to its cognate CGG-N(6)-CCG sites. We identify the uaY109 mutation, which has been shown to affect differentially the expression of a number of UaY-regulated genes, as a F112I substitution in the DNA-binding motif dimerisation domain. We isolated back mutants, revertants carrying different residues at the same position (I112N and I112M) and intragenic suppressors mapping in the first loop of the Zn cluster (N75T and N75K). We have analysed the original mutant and its revertants by growth tests and by their effects on the mRNA steady states of five UaY-regulated genes. We have determined the effect of the different mutations on UaY dimerisation, on the apparent Kdiss of the UaY DNA-binding domain to appropriate DNA sequences and on the methylation interference pattern. We have attempted to rationalise these phenotypes by modelling the UaY DNA binding domain on the structure of the highly similar Ppr1p. However, modelling of the wild-type and mutant proteins provides only a partial explanation for the observed phenotypes. This suggests that the mutated residues may have other roles besides the obvious ones inferred from their position in the sequence and by the similarity of UaY and Ppr1p.