Multiple Nuclear Localization Signals Mediate Nuclear Localization of the GATA Transcription Factor AreA

<jats:title>ABSTRACT</jats:title> <jats:p> The <jats:named-content content-type="genus-species">Aspergillus nidulans</jats:named-content> GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the <jats:named-content content-type="genus-species">Saccharomyces cerevisiae</jats:named-content> functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX <jats:sub>33</jats:sub> RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed <jats:italic>areA</jats:italic> mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from <jats:named-content content-type="genus-species">Aspergillus</jats:named-content> , yeast, and mammals, indicating an ancient origin. </jats:p>