Denning D, Mykytka B, Allen N P, Huang L, Rexach M
Cancer Biology Program, Stanford Medical School, Stanford University, CA 94305, USA.
J Cell Biol. 2001 Sep 3;154(5):937-50. doi: 10.1083/jcb.200101007.
The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.
核孔蛋白Nup60p、Nup2p和Nup1p构成了酿酒酵母核孔复合体（NPC）核篮结构的一部分。在此，我们表明这些核孔蛋白可通过在核转运蛋白Kap95p包被的珠子上进行亲和层析从酵母提取物中分离出来。为了进一步表征Nup60p，使用Nup60p包被的珠子从提取物中捕获其相互作用蛋白。我们发现Nup60p与Nup2p结合，并作为Kap95p - Kap60p异二聚体和Kap123p的对接位点。Nup60p还结合Gsp1p - GTP及其鸟嘌呤核苷酸交换因子Prp20p，并通过降低Prp20p的活性作为Gsp1p鸟嘌呤核苷酸解离抑制剂发挥作用。缺乏Nup60p的酵母在Kap60p的核输出、Kap95p - Kap60p依赖性货物的核输入以及小蛋白跨NPC的扩散方面表现出轻微缺陷。缺乏Nup60p的酵母也无法将Nup2p锚定在NPC上，导致Nup2p错误定位于核质和细胞质中。纯化的Nup60p和Nup2p直接相互结合，但当Kap60p结合Nup2p时，复合物的稳定性会受到影响。Gsp1p - GTP使Nup60p与Nup2p之间的亲和力提高10倍，并恢复Nup2p - Kap60p复合物与Nup60p的结合。结果表明在NPC处Nup60p和Nup2p之间存在由核质中Gsp1p - GTP浓度控制的动态相互作用。
