2 DISTINCT PROTEIN-PROTEIN INTERACTIONS BETWEEN THE NIT2 AND NMR REGULATORY PROTEINS ARE REQUIRED TO ESTABLISH NITROGEN METABOLITE REPRESSION IN NEUROSPORA-CRASSA

Nitrogen metabolism is a highly regulated process in Neurospora crassa . The structural genes that encode nitrogen catabolic enzymes are subj ect to nitrogen metabolite repression, mediated by the positive-acting NIT2 protein and by the negative-acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys(2)/Cys(2) zinc finger DNA-binding domain. The neg ative-acting NMR protein interacts via specific protein-protein bindin g with two distinct regions of the NIT2 protein, a short alpha-helical motif within the NIT2 DNA-binding domain and a second motif at its ca rboxy terminus. Deletions of segments of NIT2 throughout most of its l ength result in truncated proteins, which are still functional for act ivating gene expression; most of these mutant NIT2 proteins still allo w proper nitrogen repression of nitrate reductase synthesis. In contra st, deletions or certain amino acid substitutions within the zinc fing er and the carboxy-terminal tail result in a loss of nitrogen metaboli te repression. Those mutated forms of NIT2 that are insensitive to nit rogen repression have also lost one of the NIT2-NMR protein-protein in teractions. These results provide compelling evidence that the specifi c NIT2-NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.