Functional analysis of TamA, a coactivator of nitrogen-regulated gene expression in Aspergillus nidulans

The tamA gene of Aspergillus nidulans encodes a 739-amino acid protein with similarity to Uga35p/ Da181p/DurLp of Saccharomyces cerevisiae. It has bee n proposed that TamA functions as a co-activator of AreA, the major nitroge n regulatory protein in A. nidulans. Because AreA functions as a transcript ional activator under nitrogen-limiting conditions, we investigated whether TamA was also present in the nucleus. We found that a GFP-TamA fusion prot ein was predominantly localised to the nucleus in the presence and absence of ammonium, and that AreA was not required for this distribution. As the p redicted DNA-binding domain of TamA is not essential for function, we have used a number of approaches to further define functionally important region s. We have cloned the tamA gene of A. oryzae and compared its functional an d sequence characteristics with those of A. nidulans tamA and S. cerevisiae UGA35/DAL81/DURL. The Aspergillus homologues are highly conserved and func tionally interchangeable, whereas the S. cerevisiae gene does not complemen t a tamA mutant when expressed in A. nidulans. Uga35p/Da181p/DurLp was also found to be unable to recruit AreA. The sequence changes in a number of ta mA mutant alleles were determined, and altered versions of TamA were tested for tamA complementation and interaction with AreA. Changes in most region s of TamA appeared to destroy its function, suggesting that the overall con formation of the protein may be critical for its activity.