Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Abstract

SummaryNitrogen metabolism is a highly regulated process in Neurospora crassa. The structural genes that encode nitrogen catabolic enzymes are subject 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 negativeacting NMR protein interacts via specific proteinprotein binding 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 carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy-terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2-NMR protein-protein interactions. These results provide compelling evidence that the specific NIT2-NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.

A Nitrate-Induced frq-Less Oscillator in Neurospora crassa

“…The actual repression mechanism is not completely understood. Glutamine somehow activates the key regulator NMR (nitrogen metabolite regulation), which binds directly to NIT2, which is one of the two recognized positive regulators of nitrate reductase transcription (Fu and Marzluf, 1988;Marzluf, 1997;Pan et al, 1997). Thus, in the presence of only ammonium (or glutamine), the NR oscillator is not expressed, but (in frq + ) the FRQ/ WCC-driven circadian conidiation rhythm should still be operative, probably even in a nit-3 knockout mutant.…”

Nitrogen Metabolite Repression of Metabolism and Virulence in the Human Fungal Pathogen Cryptococcus neoformans

“…The Nmr proteins are inhibitors of the functions of A. nidulans AreA and N. crassa Nit2, and a potential homolog of this protein named Tar1 has recently been characterized in C. neoformans, suggesting that the regulation of GATA factor activity may operate in a similar fashion to these filamentous ascomycetes (Pan et al 1997;Andrianopoulos et al 1998;Jiang et al 2009). However, this model is confounded by conflicting observations between the studies of Jiang et al and our own.…”

“…Confusingly, Jiang et al observed growth changes on this same nitrogen source as evidence that Tar1 plays a role in nitrogen metabolism, an observation that we are unable to explain. Furthermore, C. neoformans Gat1/Are1 lacks the highly conserved C-terminus sequence of AreA and Nit2, which is involved in Nmr recognition (Platt et al 1996;Pan et al 1997;Andrianopoulos et al 1998). Nevertheless, Nmr proteins also interact with the DNA-binding domain of AreA and Nit2, and further study will be required to determine if Tar1 is a true functional ortholog of NmrA/Nmr1.…”

Lessons from the Genome Sequence ofNeurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

“…NIT-2 acts in a positive fashion to activate the expression of structural genes of diverse nitrogen pathways under conditions of nitrogen source limitation. A major negatively acting regulatory protein, NMR (for "nitrogen metabolite repression") mediates nitrogen repression and acts by directly binding to NIT-2 and inhibiting its function (590). Activation of the genes of specific pathways requires inducers and is mediated by minor control genes.…”