Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification

Abstract

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, NLStagged 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.A s regulators of retinal progenitor cell proliferation and cell specification, homeobox proteins are one set of transcription factors that are essential to vertebrate eye development (1-5). Of these, the paired-like homeobox proteins (3, 6, 7) are particularly important during retinogenesis. Vsx-1 and Vsx-2 are two paired-like homeobox proteins that were originally discovered in the regenerating visual pathway of goldfish (8 -10) and further studied in the developing zebrafish (11,12). During retinal development, the complementary expression of Vsx-1 and Vsx-2 mRNA suggests that Vsx-1 is involved in bipolar cell differentiation whereas Vsx-2 is involved in cell proliferation (12).Vsx-1 and Vsx-2 are denoted as paired-like:CVC homeobox proteins because their primary structures contains a conserved 54-aa CVC domain that is downstream and adjacent to the homeodomain. Orthologs of Vsx-1 and Vsx-2 have been isolated and characterized from other organisms, including Caenorhabditis elegans (13) and chicken (14), as well as mammals (15-17). More specifically, Chx10 is the mouse and human ortholog of Vsx-2 (18), whereas the bovine Vsx-1, named RINX͞Vsx-1, has recently been identified (16). The functional importance of these genes is clear because a human CHX10 gene mutation results in microphthalmia (19). Furthermore, a homozygous null allele for Chx10 gives rise to an ocular retardation phenotype in mice that can be partially rescued (20).Such a functional role...

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