Wu X, Kasper L H, Mantcheva R T, Mantchev G T, Springett M J, van Deursen J M
Department of Pediatrics and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3191-6. doi: 10.1073/pnas.051631598.
The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98-/- cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98-/- cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.
NUP98基因编码前体蛋白，这些前体蛋白可产生两种定位于核质的核孔蛋白，即NUP98和NUP96。通过基因靶向技术，我们选择性地破坏了小鼠的NUP98蛋白，而NUP96的表达和定位保持完整。我们发现NUP98对小鼠原肠胚形成至关重要，原肠胚形成是一个与细胞快速增殖相关的发育阶段，但对基础细胞生长则是可有可无的。NUP98基因敲除细胞具有完整的核膜，且嵌入的核孔复合体数量正常。通常，NUP98缺陷细胞的细胞质环孔板平均比对照细胞多约5倍。我们发现，一组定位于细胞质的核孔蛋白，包括NUP358、NUP214、NUP88和p62，在NUP98基因敲除细胞的核孔中组装效率低下。相反，这些核孔蛋白主要与环孔板相关。相比之下，一组定位于核质的核孔蛋白，包括NUP153、NUP50、NUP96和NUP93，对环孔板没有亲和力，并能正常组装到核孔中。突变孔在运输受体介导的具有核定位信号或M9导入信号的蛋白质对接方面显著受损，并且此类底物的核导入较弱。相比之下，突变孔导入核糖体蛋白L23a和剪接体蛋白U1A的能力似乎完好无损。这些观察结果表明，NUP98的破坏选择性地损害了离散的蛋白质导入途径，并支持了这样一种观点，即不同的导入复合体通过核孔复合体的运输是由特定的核孔蛋白亚群介导的。
