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

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.

A simple thermodynamic description of phase separation of Nup98 FG domains

“…Table 1 for a list of symbols): where R is the gas constant (8.31 J/mol·K) and T the absolute temperature in Kelvin. The G 0 term of the formula considers here that the fluorophore is (weakly) FG-philic 29 and compensates the bias from the labelling. The fit of the data revealed that one repeat unit contributes 255 J/mol to the partition equilibrium, when measured at 21°C (294 K) and 150 mM NaCl.…”

“…It was designed to match the conserved features of native Nup98 FG domains as closely as possible, including the high FG motif number/ density, compositional bias, and charge-depletion. The FG phase assembled from prf.GLFG 52x12 captures the biophysical properties and transport selectivity of the original Nup98 FG phase very well indeed 29,35 and even recapitulates RanGTPasecontrolled cargo import and export scenarios 29 . It thus represents the simplest possible experimental model of NPC-typical transport selectivity.…”

“…equipped with a 63x oil immersion objective and hybrid detectors (standard mode, in which nonlinear response of the detector was auto-corrected) at 21 ° C. Scanning settings were adjusted individually to cover wide dynamic ranges. Partition coefficient of the guest into the host (taken as signal inside FG phase: signal in buffer) was quantified as previously described 29 . prf.GLFG 52×12 [+GLEBS] was chosen as the host because of its low saturation concentration (∼0.3 μM at below 600 mM NaCl, Supp.…”

“…As an ultimate simplification, we recently engineered an FG domain (prf.GLFG 52x12 ) composed of a 52 times (perfectly) repeated 12mer GLFG peptide 29 . It was designed to match the conserved features of native Nup98 FG domains as closely as possible, including the high FG motif number/ density, compositional bias, and charge-depletion.…”

Function of the Nuclear Transport Machinery in Maintaining the Distinctive Compositions of the Nucleus and Cytoplasm

“…Conversely, the transport carrier proteins are able to penetrate the meshwork because the energy liberated from their binding to hydrophobic cores of the FG-nucleoporins is sufficient to overcome the breaking of cohesive contacts among the FG-nucleoporins [ 19 , 35 ] needed to facilitate their movement through the transport channel. Cohesive interactions among the FG-nucleoporins are thought to derive from a combination of hydrophobic, electrostatic, and other attractions, and appear to contribute to the generation of a dense meshwork within the central nuclear pore channel that impairs the movement of macromolecules [ 18 , 19 , 22 , 23 , 34 , 36 , 37 , 38 ].…”