title: Multiple nuclear localization signals mediate nuclear localization of the GATA transcription factor AreA.
abstract: The Aspergillus nidulans 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 Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA 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 Aspergillus, yeast, and mammals, indicating an ancient origin.
authors.affiliation: Department of Plant Pathology, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Cameron C
authors.initials: CC
authors.lastname: Hunter
authors.forename: Kendra S
authors.initials: KS
authors.lastname: Siebert
authors.forename: Damien J
authors.initials: DJ
authors.lastname: Downes
authors.forename: Koon Ho
authors.initials: KH
authors.lastname: Wong
authors.forename: Sara D
authors.initials: SD
authors.lastname: Kreutzberger
authors.forename: James A
authors.initials: JA
authors.lastname: Fraser
authors.forename: David F
authors.initials: DF
authors.lastname: Clarke
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hynes
authors.forename: Meryl A
authors.initials: MA
authors.lastname: Davis
authors.forename: Richard B
authors.initials: RB
authors.lastname: Todd
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: C079728:AreA protein, Aspergillus nidulans; D005656:Fungal Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D009584:Nitrogen