NmeA, a novel efflux transporter specific for nucleobases and nucleosides, contributes to metal resistance in Aspergillus nidulans

Abstract

Through Minos transposon mutagenesis we obtained A. nidulans mutants resistant to 5-fluorouracil due to insertions into the upstream region of the uncharacterized gene nmeA, encoding a Major Facilitator Superfamily (MFS) transporter. Minos transpositions increased nmeA transcription, which is otherwise extremely low under all conditions tested. To dissect the function of NmeA we used strains overexpressing or genetically lacking the nmeA gene. Strains overexpressing NmeA are resistant to toxic purine analogues, but also, to cadmium, zinc and borate, whereas an isogenic nmeAΔ null mutant exhibits increased sensitivity to these compounds. We provide direct evidence that nmeA overexpression leads to efflux of adenine, xanthine, uric acid and allantoin, the latter two being intermediate metabolites of purine catabolism that are toxic when accumulated cytoplasmically due to relevant genetic lesions. By using a functional GFP-tagged version we show that NmeA is a plasma membrane transporter. Homology modeling and docking approaches identified a single purine binding site and a tentative substrate translocation trajectory in NmeA. Orthologues of NmeA are present in all Aspergilli and other Eurotiomycetes, but are absent from other fungi or non-fungal organisms. NmeA is thus the founding member of a new class of transporters essential for fungal success under specific toxic conditions.

Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans

“…These include proteins that might be involved in the export of aflatoxin (AN5370/AN7200), fluconazole (AN5329/AN4119/AN8089), aspyridone polyketide (AN8413), antibiotics (AN8366) and drugs (AN4481, AN1031, AN11217). Still, other MFS, also tentatively associated to the efflux of similar toxic metabolites (AN0970, AN7295, AN0018, AN2643, AN3254, AN9219, AN9382, AN7898, AN2840), or NmeA/AN2746 associated to efflux of toxic amounts of nucleobases and Cd [ 30 ], were not detected in our analysis. Finally, we detected several known major siderophore efflux transporters, such as MirA, MirB, and MirC [ 31 ].…”

Mycoextraction: Rapid Cadmium Removal by Macrofungi-Based Technology from Alkaline Soil

“…MFS is the largest transporter group, which might mediate the transport of relevant secondary metabolites across membranes via facilitated diffusion, symport, or antiport [50]. It is difficult to predict whether an MFS is acting as an importer or an exporter [51]. Some MFS genes could increase Zn and Cd tolerance, through metal sequestration to vacuoles [52] or efflux [51].…”

“…It is difficult to predict whether an MFS is acting as an importer or an exporter [51]. Some MFS genes could increase Zn and Cd tolerance, through metal sequestration to vacuoles [52] or efflux [51]. Transporters for Ca 2+ /H + , K + and Na + were also found to be upregulated significantly.…”