Functional characterization of tzn1 and tzn2-zinc transporter genes in Neurospora crassa

Previous work from our laboratory involved the description of the Neurospora metal transportome, which included seven hypothetical zinc transporters belonging to the ZIP family. The aim of the present study was to make a comparative functional evaluation of two hypothetical zinc transporters named tzn1 (NCU07621.3) and tzn2 (NCU11414.3). Phenotypic analysis of tzn1 and tzn2 mutants and a double mutant (tzn1tzn2) revealed that the deletion of tzn1 causes aconidiation and a greater defect in growth than the single deletion of tzn2. Supplementation with zinc restores growth but not conidiation in tzn1 and tzn1tzn2. TZN1 complemented a zinc-uptake-deficient Saccharomyces cerevisiae mutant (zrt1zrt2) in zinc-deficient conditions, while tzn2 restored growth upon supplementation with zinc (0.05 mM). Furthermore, the Deltatzn1 mutant was found to have severely reduced zinc content indicating that tzn1 functions as a key regulator of intracellular zinc levels in Neurospora crassa. Zinc uptake studies indicate tzn1 is a specific transporter of zinc, while tzn2 transports both zinc and cadmium. Quantitative RT-PCR showed up-regulation of tzn1 (128-fold) under zinc-depleted conditions and down-regulation (>1,000-fold) in zinc-replete conditions. The present study indicates that the zinc transport proteins encoded by tzn1 and tzn2 are members of the zinc uptake system regulated by zinc status in N. crassa.