title: Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358.
abstract: 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.
authors.affiliation: The Picower Institute for Medical Research, Manhasset, New York 11030, USA. hisaito@molbiol.med.kyushu-u.ac.jp
authors.forename: Hisato
authors.initials: H
authors.lastname: Saitoh
authors.forename: Maryann Delli
authors.initials: MD
authors.lastname: Pizzi
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
mesh_terms: D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016685:Mitomycin / Q000494:pharmacology; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051135:Rad51 Recombinase; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes; D014981:Xenopus; D029867:Xenopus Proteins; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D029867:Xenopus Proteins; C094493:ran-binding protein 2; D049452:Green Fluorescent Proteins; D016685:Mitomycin; D005047:Etoposide; D044763:Ubiquitin-Conjugating Enzymes; D005982:Glutathione Transferase; C495152:RAD51 protein, Xenopus; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9