Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies.
Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies.
复制标题
FUS 和 Nup62 C 端结构域之间的相互作用足以使其共相分离成无定形组装体。
DOI：
10.1016/j.jmb.2023.167972
复制
发表时间：
2023
影响因子：
5.6
通讯作者：
Bosco,DarylA
中科院分区：
文献类型：
--
作者：
Kumar,MeenakshiSundaram;Stallworth,KarlyM;Murthy,AnastasiaC;Lim,SuMin;Li,Nan;Jain,Aastha;Munro,JamesB;Fawzi,NicolasL;Lagier-Tourenne,Clotilde;Bosco,DarylA
核质转运缺陷是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的一个致病特征，包括融合肉瘤（FUS）突变引起的ALS。最近，野生型和ALS连锁的突变体FUS都显示出与富含苯丙氨酸-甘氨酸（FG）的核孔蛋白62（Nup 62）蛋白直接相互作用，其中FUS WT/Nup 62相互作用在细胞核内富集，但ALS连锁的突变体FUS/Nup 62相互作用在细胞质内富集。Nup 62是一个中央通道Nup，在形成核孔复合物内的选择性过滤器和调节有效的核质转运中具有突出的作用。在FUS相在体外分离成液滴的条件下，Nup 62的加入引起含有FUS和Nup 62的无定形组装体的协同形成。在这里，我们研究了这个过程中使用重组FUS和Nup 62蛋白和生化方法的分子决定因素。我们证明，Nup 62的结构化的C-末端结构域包含一个α-螺旋卷曲螺旋区域在结合FUS中起着主导作用，并且足以诱导FUS/Nup 62无定形组装体的形成。相比之下，Nup 62的天然非结构化的、富含F/G重复的N-末端结构域适度地促成FUS/Nup 62相分离行为。单个Nup 62结构域构建体在人细胞中的表达证实了Nup 62 C-末端结构域对于蛋白质定位于核膜是必需的。我们的研究结果提高了FUS和Nup 62的C-末端结构域之间的相互作用可以影响Nup 62在生理和/或病理条件下的功能的可能性。
Deficient nucleocytoplasmic transport is emerging as a pathogenic feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including in ALS caused by mutations in Fused in Sarcoma (FUS). Recently, both wild-type and ALS-linked mutant FUS were shown to directly interact with the phenylalanine-glycine (FG)-rich nucleoporin 62 (Nup62) protein, where FUS WT/ Nup62 interactions were enriched within the nucleus but ALS-linked mutant FUS/ Nup62 interactions were enriched within the cytoplasm of cells. Nup62 is a central channel Nup that has a prominent role in forming the selectivity filter within the nuclear pore complex and in regulating effective nucleocytoplasmic transport. Under conditions where FUS phase separates into liquid dropletsin vitro, the addition of Nup62 caused the synergistic formation of amorphous assemblies containing both FUS and Nup62. Here, we examined the molecular determinants of this process using recombinant FUS and Nup62 proteins and biochemical approaches. We demonstrate that the structured C-terminal domain of Nup62 containing an alpha-helical coiled-coil region plays a dominant role in binding FUS and is sufficient for inducing the formation of FUS/Nup62 amorphous assemblies. In contrast, the natively unstructured, F/G repeat-rich N-terminal domain of Nup62 modestly contributed to FUS/Nup62 phase separation behavior. Expression of individual Nup62 domain constructs in human cells confirmed that the Nup62 C-terminal domain is essential for localization of the protein to the nuclear envelope. Our results raise the possibility that interactions between FUS and the C-terminal domain of Nup62 can influence the function of Nup62 under physiological and/or pathological conditions.