Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG repeats in NPC are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated nucleocytoplasmic transport of proteins in both directions, and decreasing modification slowed transport. Super-resolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.

中文翻译：

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O-GlcNAc修饰核孔复合体可加速双向转运

跨越核膜的大分子运输取决于通过核孔复合物（NPC）的促进扩散。NPC的内部包含由苯丙氨酸-甘氨酸（FG）重复域构成的渗透屏障，可选择性地促进与核转运受体（NTR）结合的货物的渗透。NPC中的FG重复序列是O-连接的N-乙酰氨基葡萄糖（O-GlcNAc）修饰的主要位点，但这种修饰在核质转运中的功能作用尚不清楚。我们开发了基于光遗传学探针的高通量测定法，以量化活细胞中核导入和导出的动力学。我们发现增加NPC的O-GlcNAc修饰会加快NTR促进蛋白质在两个方向上的核质转运，而减少修饰会减慢转运。超分辨率成像显示在FG重复屏障处O-GlcNAc的富集程度很高。O-GlcNAc修饰还可以加速小分子惰性蛋白质通过NPC的被动渗透。我们得出的结论是，O-GlcNAc修饰可通过增强FG重复屏障的非特异性通透性来加速核质运输，也许是通过空间抑制FG重复序列之间的相互作用来实现。