Cloning of karyopherin-alpha3 from Drosophila through its interaction with the nuclear localization sequence of germ cell-less protein.
The D. melanogaster germ cell-less (gcl) gene has previously been shown to play a key role in the establishment of the germ cell lineage during fly embryogenesis. To identify other molecules that function with Gcl in this process, we have conducted a yeast two-hybrid screen that utilized Gcl protein as bait. A predominant class of Gcl-interacting clones encodes a species of importin-alpha from Drosophila (karyopherin-alpha3; kap-alpha3), a nuclear-localization sequence binding protein previously shown to act in the transport of proteins from the cytoplasm to the nucleus. The expression of kap-alpha3 is widespread both temporally and spatially throughout the embryo during development, as judged by Northern blotting and whole-mount in situ hybridization to Drosophila embryos, suggesting that it functions at multiple stages of development. Studies of the Gcl/Kap-alpha3 interaction have identified a functional nuclear-localization sequence in Gcl protein which is necessary for an in vivo interaction and for nuclear entry of Gcl, making it likely that one role for Kap-alpha3 is to deliver Gcl protein to the nucleus. The identification of Kap-alpha3 and an in vivo substrate will allow for further characterization of the basis for specificity between importin-alpha molecules and their binding substrates.
Dockendorff
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Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia 19104-6100, USA.
Tang
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Jongens
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D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics; D003001:Cloning, Molecular; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D028884:Karyopherins; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D028901:alpha Karyopherins
D002352:Carrier Proteins; D029721:Drosophila Proteins; C471550:Gcl protein, mouse; D019476:Insect Proteins; D036341:Intercellular Signaling Peptides and Proteins; C106306:KPNA3 protein, human; D028884:Karyopherins; D009687:Nuclear Proteins; D028901:alpha Karyopherins; C076187:gcl protein, Drosophila