Assembly principle of a membrane-anchored nuclear pore basket scaffold.

Nuclear pore complexes (NPCs) are membrane-embedded gatekeepers of traffic between the nucleus and cytoplasm. Key features of the NPC symmetric core have been elucidated, but little is known about the NPC basket, a prominent structure with numerous roles in gene expression. Studying the basket was hampered by its instability and connection to the inner nuclear membrane (INM). Here, we reveal the assembly principle of the yeast NPC basket by reconstituting a recombinant Nup60-Mlp1-Nup2 scaffold on a synthetic membrane. Nup60 serves as the basket's flexible suspension cable, harboring an array of short linear motifs (SLiMs). These bind multivalently to the INM, the coiled-coil protein Mlp1, the FG-nucleoporin Nup2, and the NPC core. We suggest that SLiMs, embedded in disordered regions, allow the basket to adapt its structure in response to bulky cargo and changes in gene expression. Our study opens avenues for the higher-order reconstitution of basket-anchored NPC assemblies on membranes.

Phase separation directs ubiquitination of gene-body nucleosomes.

The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.