title: Overlap of nuclear localisation signal and specific DNA-binding residues within the zinc finger domain of PacC.
abstract: The transcription factor PacC, mediating regulation of gene expression by ambient pH in the genetically amenable fungus Aspergillus nidulans, contains a three zinc finger DNA-binding domain (zf-DBD) including a nuclear localisation signal (NLS). We selected 38 novel mutations impairing PacC function, of which 21 missense mutations identify individual residues essential for zf-DBD structure/function. Our functional analysis agrees with our previous conclusion that finger 1 does not bind DNA and provides in vivo evidence that Trp80 and Trp116, located in the Cys knuckles of adjacent zinc fingers, are critical for zf-DBD structure/function. In the finger 3 alpha-helix, Gln155 (+4) is specifically involved in contacting DNA, while the major role of Lys159 (+6) resides in the nuclear localisation of the protein. In contrast, Lys158 is essential for DNA binding and for nuclear localisation. As finger 3 suffices to drive nuclear localisation of green fluorescent protein, we conclude that it contains an NLS including essential Lys158 and Lys159. These residues are within an alpha-helical basic sequence that is completely conserved amongst zinc fingers of the PacC/RIM101 family and present in an identical position of the last finger alpha-helix of Drosophila Cubitus interruptus, where it is also involved in nuclear localisation. We propose that PacC and Gli/Ci zf-DBDs belong to a subclass of these domains characterised by possession of a pair of conserved Trp residues involved in the interaction between the two most N-terminal fingers and the presence of an NLS in the alpha-helix of the most C-terminal finger. Loss of PacC nuclear localisation resulting from His142Leu (beta-strand) and Phe151Ser (hydrophobic core) substitutions in finger 3 suggests that its folding is required for NLS function. Overlap of DNA binding and NLS may aid release of PacC from its cognate importer(s) upon nuclear translocation, as suggested for zinc binuclear cluster proteins.
authors.affiliation: Departamento de Microbiologia Molecular, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu, 9, Madrid 28040, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Fernández-Martínez
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Brown
authors.forename: Eliecer
authors.initials: E
authors.lastname: Díez
authors.forename: Joan
authors.initials: J
authors.lastname: Tilburn
authors.forename: Herbert N
authors.initials: HN
authors.lastname: Arst
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Peñalva
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Espeso
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals; D010641:Phenotype; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016335:Zinc Fingers; D028901:alpha Karyopherins / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D019913:Nuclear Localization Signals; C092394:PacC protein, Aspergillus; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D028901:alpha Karyopherins; D005680:gamma-Aminobutyric Acid