title: Identification of a classical bipartite nuclear localization signal in the Drosophila TEA/ATTS protein scalloped.
abstract: Drosophila melanogaster wing development has been shown to rely on the activity of a complex of two proteins, Scalloped (Sd) and Vestigial (Vg). Within this complex, Sd is known to provide DNA binding though its TEA/ATTS domain, while Vg modulates this binding and provides transcriptional activation through N- and C-terminal activation domains. There is also evidence that Sd is required for the nuclear translocation of Vg. Indeed, a candidate sequence which shows consensus to the bipartite family of nuclear localization signals (NLSs) has been identified within Sd previously, though it is not known if it is functional, or if additional unpredicted signals that mediate nuclear transport exist within the protein. By expressing various enhanced green fluorescent protein (eGFP) tagged constructs within Drosophila S2 cells, we demonstrate that this NLS is indeed functional and necessary for the proper nuclear localization of Sd. Additionally, the region containing the NLS is critical for the wildtype function of ectopically expressed Sd, in the context of wing development. Using site-directed mutagenesis, we have identified a group of five amino acids within this NLS which is critical for its function, as well as another group of two which is of lesser importance. Together with data that suggests that this sequence mediates interactions with Importin-α3, we conclude that the identified NLS is likely a classical bipartite signal. Further dissection of Sd has also revealed that a large portion of the C-terminal domain of the protein is required its proper nuclear localization. Finally, a Leptomycin B (LB) sensitive signal which appears to facilitate nuclear export is identified, raising the possibility that Sd also contains a nuclear export signal (NES).
authors: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
authors: Adam C
authors: AC
authors: Magico
authors: John B
authors: JB
authors: Bell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005799:Genes, Dominant; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014921:Wings, Animal / Q000187:drug effects / Q000254:growth & development
chemical_list: D029721:Drosophila Proteins; D005231:Fatty Acids, Unsaturated; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; C087984:sd protein, Drosophila; D049452:Green Fluorescent Proteins; C038753:leptomycin B