Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification
Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification
复制标题
Ubc9 与核定位信号相互作用并介导成对同源染色体蛋白 Vsx-1 的核定位，与 SUMO-1 修饰无关
DOI：
10.1073/pnas.101129698
复制
发表时间：
2001-05-08
影响因子：
11.1
通讯作者：
Schechter, N
中科院分区：
文献类型：
--
作者：
Kurtzman, AL;Schechter, N
Vsx-1是一个配对样：CVC同源框基因，其表达与斑马鱼视网膜发生过程中的双极细胞分化有关。我们使用酵母双杂交筛选来鉴定与Vsx-1和分离的Ubc 9相互作用的蛋白质，Ubc 9是一种缀合小泛素样修饰物SUMO-1的酶。尽管它与Ubc 9相互作用，但我们表明Vsx-1不是COS-7细胞或体外SUMO-1的底物。对Vsx-1相互作用结构域的缺失分析表明，Ubc 9在同源结构域的NH 2末端结合核定位信号（NLS）。在SW 13细胞中，Vsx-1定位于细胞核，并被排除在核仁之外。NLS的缺失破坏了这种核定位，导致Vsx-1的弥漫性胞质分布。在表达低水平内源性Ubc 9的SW 13 AK 1细胞中，Vsx-1积聚在核周环中并与内质网标记物共定位。然而，NLS标记的STAT 1蛋白在SW 13和SW 13 AK 1细胞中都表现出正常的核定位，表明核输入没有被全面破坏。Vsx-1与Ubc 9的共转染恢复了SW 3 AK 1细胞中Vsx-1的核定位，并证明Ubc 9是Vsx-1的核定位所需的。Ubc 9继续恢复核定位，即使在C935活性位点突变消除了其SUMO-1缀合能力之后。这些结果表明，Ubc 9通过非酶机制介导Vsx-1和可能的其他蛋白质的核定位，该机制不依赖于SUMO-1缀合，
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 NH2 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 C935 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,