Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification.
Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH(2) terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.
Kurtzman
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AL
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794, USA.
Schechter
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N
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D025842:SUMO-1 Protein; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D005136:Eye Proteins; D018398:Homeodomain Proteins; D019913:Nuclear Localization Signals; D025842:SUMO-1 Protein; D014452:Ubiquitins; C491583:VSX1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9