Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358.
Ubc9, a conjugation enzyme for the ubiquitin-related modifier SUMO, is present predominantly in the nucleus and at the nuclear pore complex. The functional significance of its subcellular compartmentalization, however, remains to be elucidated. Here, we define a Pro-Glu-Asp-Ser-Thr-rich element containing 129 amino acid residues, designated IR1+2, on the human nucleoporin ... RanBP2/Nup358, which binds directly to Ubc9 with high affinity both in vitro and in vivo. When IR1+2 tagged with green fluorescence protein at its amino terminus (GFP-IR1+2) was transfected into COS-7 cells, we found that approximately 90% of the nuclear Ubc9 was sequestered in the cytoplasm. We also observed that both SUMO-1 and SUMO-2/3 were mislocalized, and promyelocytic leukemia protein PML formed an enlarged aggregate in the nucleus. Moreover, the homologous recombination protein Rad51 mislocalized to the cytoplasm, and Rad51 foci, a hallmark of functional association of Rad51 with damaged DNA, did not form efficiently even in the presence of a DNA strand breaker. These findings emphasize that the IR1+2 domain is a useful tool for manipulating the nuclear localization of Ubc9 and perturbing the subcellular localization of SUMOs and/or SUMOlated proteins, and they emphasize the important role of nuclear Ubc9 in the Rad51-mediated homologous recombination pathway, possibly by modulating intracellular trafficking of Rad51.
Mesh Terms:
Active Transport, Cell Nucleus, Amino Acid Motifs, Amino Acid Sequence, Animals, COS Cells, Cell Line, Cell Nucleus, Cricetinae, Cytoplasm, DNA-Binding Proteins, Etoposide, Fluorescent Antibody Technique, Indirect, Glutathione Transferase, Green Fluorescent Proteins, Humans, Ligases, Luminescent Proteins, Microscopy, Fluorescence, Mitomycin, Molecular Chaperones, Molecular Sequence Data, Nuclear Pore Complex Proteins, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Protein Transport, Rad51 Recombinase, Recombinant Fusion Proteins, Recombination, Genetic, Small Ubiquitin-Related Modifier Proteins, Transfection, Ubiquitin-Conjugating Enzymes, Xenopus, Xenopus Proteins
Active Transport, Cell Nucleus, Amino Acid Motifs, Amino Acid Sequence, Animals, COS Cells, Cell Line, Cell Nucleus, Cricetinae, Cytoplasm, DNA-Binding Proteins, Etoposide, Fluorescent Antibody Technique, Indirect, Glutathione Transferase, Green Fluorescent Proteins, Humans, Ligases, Luminescent Proteins, Microscopy, Fluorescence, Mitomycin, Molecular Chaperones, Molecular Sequence Data, Nuclear Pore Complex Proteins, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Protein Transport, Rad51 Recombinase, Recombinant Fusion Proteins, Recombination, Genetic, Small Ubiquitin-Related Modifier Proteins, Transfection, Ubiquitin-Conjugating Enzymes, Xenopus, Xenopus Proteins
J. Biol. Chem.
Date: Feb. 15, 2002
PubMed ID: 11709548
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