Evidence for post‐translational regulation of NrtA, the Aspergillus nidulans high‐affinity nitrate transporter

Abstract

Summary• Here, influx and efflux of , and net fluxes of and , were measured in Aspergillus nidulans mutants niaD171 and niiA5 , devoid of nitrate reductase (NR) and nitrite reductase (NiR) activities, respectively.• Transcript and protein abundances of NrtA, the A. nidulans principal high-affinity transporter, were determined using semiquantitative reverse transcriptionpolymerase chain reaction and western blots, respectively.• influx in niaD171 was negligible relative to wild-type values, whereas efflux to influx ratios increased nine-fold. Nevertheless, NrtA mRNA and NrtA protein were expressed at levels more than two-fold and three-fold higher, respectively, in niaD171 than in the wild-type strain.• This is the first demonstration of diminished high-affinity influx associated with elevated transporter levels, providing evidence that, in addition to transcriptional regulation, control of NrtA expression operates at the post-translational level. This mechanism allows for rapid control of transport at the protein level, reduces the extent of futile cycling of that would otherwise represent a significant energy drain when influx exceeds the capacity for assimilation or storage, and may be responsible for the rapid switching between the on and off state that is associated with simultaneous provision of to mycelia absorbing .

Nitrite transport is mediated by the nitrite-specific high-affinity NitA transporter and by nitrate transporters NrtA, NrtB in Aspergillus nidulans

“…One possibility is that its primary responsibility is for NO 2 À efflux under conditions where NO 3 À uptake and reduction exceed the organism's capacity for further assimilation. It is well documented that when NO 3 À uptake and reduction exceed capacity for reduction to NH 4 + , NO 2 À is excreted (Collos, 1998;Jia and Cole, 2005;Wang et al, 2007). Disruption of the nitA gene, in the double mutant background in the present study, reduced NO 2 À uptake to very low rates that were pHdependent in a manner suggestive of passive permeation in the form of HNO 2 .…”

“…In order to measure nitrite efflux samples of 13 NO 3 were reduced to 13 NO 2 À by use of a cadmium column following the method of McElfresh et al (1979). The double mutant, strain nrtA747 nrtB110, was incubated in standard influx medium containing 500 lM 13 NO 2 À for 30 min and then the labelled medium was replaced by identical non-labelled medium as described in Wang et al (2007) for the determination of 13 N efflux. Non-labelled media were replaced at intervals of 1 min (five times) and 2 min (eight times) for a total of 21 min and the eluates counted to detect tracer leaving the mycelia.…”

“…Using the standard calculations (Wang et al, 2007), 13 NO 2 À efflux values were determined to be 5.3 nmol mg À1 DW h À1 . This is approximately 5% of influx.…”

Npr1 Ser/Thr Protein Kinase Links Nitrogen Source Quality and Carbon Availability with the Yeast Nitrate Transporter (Ynt1) Levels

“…However, when nitrate is scarce, its fine regulation by Npr1-mediated phosphorylation targets it to the plasma membrane, facilitating nitrate uptake and consequently nitrate assimilation gene induction (12). There is also strong evidence on the post-translational regulation of A. thaliana NRT2.1 and Aspergillus nidulans NrtA (13,14).…”

Phosphorylation of the Yeast Nitrate Transporter Ynt1 Is Essential for Delivery to the Plasma Membrane during Nitrogen Limitation

“…Interestingly, nitrogen limitation renders the lowest levels of Ynt1 ubiquitinylation, and consequently it accumulates at the plasma membrane. More recently, it has been suggested that nitrate transporters NrtA and NRT2.1 from Aspergillus nidulans and Arabidopsis thaliana undergo post-translational regulation but the molecular mechanisms are so far unknown (7,14,15).…”