Nuclear localization of the Hermes transposase depends on basic amino acid residues at the N-terminus of the protein.
For the Hermes transposable element to be mobilized in its eukaryotic host, the transposase, encoded by the element, must make contact with its DNA. After synthesis in the cytoplasm, the transposase has to be actively imported into the nucleus because its size of 70.1 kDa prevents passive diffusion through the nuclear pore. Studies in vitro using transient expression of a Hermes-EGFP fusion protein in Drosophila melanogaster Schneider 2 cells showed the transposase was located predominantly in the nucleus. In silico sequence analysis, however, did not reveal any nuclear localization signal (NLS). To identify the sequence(s) responsible for localization of Hermes transposase in the nucleus, truncated or mutated forms of the transposase were examined for their influence on sub-cellular localization of marker proteins fused to the transposase. Using the same expression system and a GFP-GUS fusion double marker, residues 1-110 were recognized as sufficient, and residues 1-32 as necessary, for nuclear localization. Amino acid K25 greatly facilitated nuclear localization, indicating that at least this basic amino acid plays a significant role in this process. This sequence overlaps the proposed DNA binding region of the Hermes transposase and is not necessarily conserved in all members of the hAT transposable element family.
Michel
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Department of Entomology, University of California, Riverside, California 92521, USA.
Atkinson
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PW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D004251:DNA Transposable Elements / Q000502:physiology; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics; D017353:Gene Deletion; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019895:Transposases; D005966:Glucuronidase; D008239:Lysine