Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity

Abstract

Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic ‘FG particles’. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTR⋅cargo complexes, while cargo shielding and increased NTR⋅cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore.

DOI: http://dx.doi.org/10.7554/eLife.04251.001

eLife digest

Cells of eukaryotic species—which include plants, animals, and fungi—have a nucleus that harbours the organism's genome. Two membrane layers surround the nucleus and separate its contents from the cytoplasm, where proteins are made. This separation is essential for a correct interpretation of the genetic information. Yet, various molecules, such as proteins, need to move into or out of the nucleus for the cell to work properly. This transit has to occur without an uncontrolled mixing of the contents of the nucleus and the cytoplasm happening.

Structures called nuclear pore complexes span the double membrane and allow material to be exchanged between the nucleus and the cytoplasm. Small molecules can freely pass through these complexes, while larger molecules can only be transported when bound as “cargo” to so-called nuclear transport receptors. Nuclear pore complexes are large assemblies of proteins called nucleoporins. FG nucleoporins are special in that they contain regions with a repeating pattern of two amino acids, phenylalanine (‘F’) and glycine (‘G’). These regions are called FG domains. They bind to nuclear transport receptors and have been suspected to form a barrier that decides which molecules may pass through the nuclear pore complex. Exactly how this control is exerted has been a matter of debate.

Versions of a particular FG nucleoporin called Nup98 are found in all branches of eukaryotic life, i.e. in animals, fungi, plants, amoebas, and even in the evolutionarily most distant protozoans. When Schmidt and Görlich dispersed small amounts of Nup98 FG domains in an aqueous solution, the domains rapidly attracted each other to form ‘FG particles’, regardless of which species the proteins came from. These FG particles were so dense that they repelled ‘normal’ macromolecules, yet they allowed nuclear transport receptors, along with their bound cargoes, to rapidly enter. Taken together, the work of Schmidt and Görlich suggests that such FG particles form the transport barrier in nuclear pore complexes.

Based on these findings, Schmidt and Görlich refine a model where the FG domains form a mesh in the nuclear pore complexes that acts like a ‘smart sieve’. Smaller molecules can move through gaps in the meshwork, but larger molecules are hindered. Schmidt and Görlich suggest that nuclear transport receptors help large molecules to move through nuclear pore complexes by ‘melting’ the FG meshwork locally, creating a path for the molecule to move through. The reconstitution of these smart barriers in the laboratory will now allow researchers to analyse the process of receptor-mediated nuclear pore passage in unprecedented (mechanistic) detail.

DOI: http://dx.doi.org/10.7554/eLife.04251.002