Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide

Sheung Chun Ng,Dirk Görlich,Thomas Güttler

doi:10.1038/s41467-021-24292-5

Sheung Chun Ng, Dirk Görlich + Show 1 more

Open Access

https://doi.org/10.1038/s41467-021-24292-5

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Abstract

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. The barrier can be described as a condensed phase assembled from cohesive FG repeat domains. NPCs contain several distinct FG domains, each comprising variable repeats. Nevertheless, we now found that sequence heterogeneity is no fundamental requirement for barrier function. Instead, we succeeded in engineering a perfectly repeated 12mer GLFG peptide that self-assembles into a barrier of exquisite transport selectivity and fast transport kinetics. This barrier recapitulates RanGTPase-controlled importin- and exportin-mediated cargo transport and thus represents an ultimately simplified experimental model system. An alternative proline-free sequence forms an amyloid FG phase. Finally, we discovered that FG phases stain bright with ‘DNA-specific’ DAPI/ Hoechst probes, and that such dyes allow for a photo-induced block of nuclear transport.

Highlights

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport
Importins and exportins are capable of active transport against concentration gradients, drawing energy from the nucleocytoplasmic RanGTP gradient[8]
Importins capture cargo at low RanGTP-levels in the cytoplasm, translocate through NPCs, release their cargo when encountering RanGTP inside nuclei, and return as importin·RanGTP complexes to the cytoplasm

Summary

Introduction

The permeability barrier of nuclear pore complexes (NPCs) controls nucleocytoplasmic transport. It retains inert macromolecules while allowing facilitated passage of importins and exportins, which in turn shuttle cargo into or out of cell nuclei. Nuclear pore complexes (NPCs) perforate the nuclear envelope and enable nucleocytoplasmic exchange through a 70-nm wide central channel[1,2,3,4] Passage through this central channel is controlled by a sieve-like permeability barrier that grants rapid passage to small molecules but becomes increasingly restrictive as the size of the mobile species approaches or exceeds a limit of ≈30 kDa5. The sieve effect of such dense FG phases excludes “inert” macromolecules, yet the “FGphilic” NTRs can rapidly enter and traverse them[6,30,32]

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