PMC

Simplified secondary structure model of the high-affinity nitrate transporter NtrA of A. nidulans. Positions of residues R87, N168, E330, R368, and N459 are shown, with the two MFS motifs indicated and the two nitrate signatures marked by hatched boxes.

Expression of NrtA. Approximately 50 μg of protein from membrane fractions of strains carrying single copies of the V5-tagged constructs was subjected to 10% SDS/PAGE followed by immunoblot analysis by using anti-V5-horseradish peroxidase monoclonal antibody. V5WT refers to the wild-type NrtA construct with a C-terminal V5 epitope tag, and WT refers to the wild-type construct without the V5 tag. Mutant constructs are indicated above the respective lanes. Immunoblot data for mutants not shown can be viewed upon request. Details of mutant strains are given for or in the text.

Growth tests of single-copy nrtA gene transformants. Shown are representative single colonies of arginine prototrophic transformants of JK1060 grown on minimal medium containing 10 mM nitrate as the sole nitrogen source. Transformant strains R87K T2, R368K T8, and N168Q T12, each possess a single nrtA mutant copy located at argB. Transformant JK1060-pILJ16 (a vector that contains the argB gene only) is used as the control for the base level of growth on nitrate. Transformant T7920 is a single-copy integration at argB of the wild-type coding region in pMUT (containing the wild-type nrtA gene) and was included as a control for wild-type growth. R87T/N459K is the intragenic suppressor mutant generated by chemical mutagenesis of a single-copy R87T T1 strain.