The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion

Abstract

Nuclear pore complexes (NPCs) restrict the nucleocytoplasmic flux of most macromolecules, but permit facilitated passage of nuclear transport receptors and their cargo complexes. We found that a simple hydrophobic interaction column can mimic the selectivity of NPCs surprisingly well and that nuclear transport receptors appear to be the most hydrophobic soluble proteins. This suggests that surface hydrophobicity represents a major sorting criterion of NPCs. The rate of NPC passage of cargo-receptor complexes is, however, not dominated just by properties of the receptors. We found that large cargo domains drastically hinder NPC passage and require more than one receptor molecule for rapid translocation. This argues against a rigid translocation channel and instead suggests that NPC passage involves a partitioning of the entire translocating species into a hydrophobic phase, whereby the receptor:cargo ratio determines the solubility in that permeability barrier. Finally, we show that interfering with hydrophobic interactions causes a reversible collapse of the permeability barrier of NPCs, which is consistent with the assumption that the barrier is formed by phenylalanine-rich nucleoporin repeats that attract each other through hydrophobic interactions.

Fig. 1. Surface hydrophobicity is one marker for translocation competence. A cytosolic extract from HeLa cells was prepared and subjected to binding to immobilized RanGDP, RanGTP or phenyl-Sepharose (low substitution). Analysis of starting material and bound fractions was by SDS–PAGE followed by Coomassie Blue staining (left panel) or western blotting with antibodies raised against the indicated nuclear transport receptors (right panels). Phenyl-Sepharose retrieved Impβ transport receptors with specificity similar to that of RanGTP. Certain receptors (e.g. Imp9) were recovered with even higher efficiency by phenyl-Sepharose than by RanGTP. Likewise, NTF2 bound phenyl-Sepharose as efficiently as its import substrate RanGDP. In contrast, typical cytosolic proteins, such as eIF-5A, did not bind to the hydrophobic matrix under these stringent conditions (see also Materials and methods and main text). Load in the bound fraction corresponds to 20× the starting material.

Fig. 2. Two scenarios for facilitated NPC passage. (A) The receptor contacts Phe-rich motifs (red dots) at the periphery of the channel, which triggers a movement through the otherwise empty (i.e. purely aqueous) and rigid channel. (B) In the second scenario, receptor–cargo complexes must partition completely into a tightly sealing selective phase, which implies that the receptor (which is attracted by the phase) as well as the cargo (which is repelled by the phase) become exposed to the permeability barrier.

Fig. 3. Inert cargo domains hinder NPC passage of Ran. Ran was expressed with three different N-terminal tags in order to obtain Ran derivatives of increasing size. These tags were a his tag, a zz domain (IgG binding domain from protein A) or MBP. Nuclear import of the Alexa 488-labelled fusion proteins (1 µM each) was performed in the presence of NTF2 (one homodimer per Ran molecule) and an energy regenerating system (see Materials and methods). The nucleocytoplasmic Ran distribution was recorded in real time by confocal scans through the import mixtures. Quantitation of the data revealed that influx of zzRan and MBP–Ran occurred 3-fold and 15-fold, respectively, more slowly than influx of his-tagged Ran. The 5 min time point is a control to demonstrate that MBP–Ran is capable of nuclear accumulation even though its influx is slow.

Fig. 4. (A) The rate of facilitated translocation is determined not only by properties of the receptor, but also by those of the cargo. Three different inert proteins, namely GFP, MBP and an MBP dimer (2×MBP), were each fused to an IBB domain (a potent Impβ-dependent import signal). The GFP fusion was detected through its intrinsic fluorescence. Fluorescent MBP fusion proteins had been labelled with Alexa 488 maleimide. The fluorescent fusion proteins (0.5 µM final) were pre-bound to stoichiometric amounts of Impβ and their import into nuclei of permeabilized cells was allowed in the presence of Ran and an energy-regenerating system. The distribution of the fusions was determined by confocal fluorescence microscopy. Panels show the reactions after 9 s of import. Even though the three substrates were linked to identical import signals and imported under identical conditions by the same receptor, their import rates were strikingly different. Thus, the translocation rate is not only determined by the receptor and the type of signal, but also by the ‘rest’ of the cargo domain. (B) Enlarged field of the IBB–2×MBP sample from (A). Clear NPC staining is evident even against the high background of cytoplasmic substrate.

Fig. 5. Large cargoes can cross NPCs rapidly, provided that more than one receptor is recruited. The fluorescent import substrate (0.4 µM) contains an N-terminal IBB domain for Impβ-dependent import, a double MBP cargo segment and a C-terminal M3 domain for transportin-mediated import. Import was performed with Ran, an energy-regenerating system and the indicated combinations of transportin and Impβ. Receptor:cargo stoichiometry is indicated. As the permeabilized cells contain significant amounts of endogenous transportin and Impβ, endogenous receptors had to be quenched with either 2 µM unlabelled MBP–M3 (minus transportin samples) or IBB–MBP (minus Impβ samples). Transportin and Impβ show a high degree of cooperativity in the import of this fusion protein. Relative influx rates were as follows: 0.28 with Impβ, 0.18 with transportin and 3.2 with Impβ plus transportin. The 2× Impβ and 2× transportin control reactions verified that the added transportin or Impβ was not limiting for the import reactions and that further increase in their concentration did not improve import.

Fig. 6. Interference with hydrophobic interactions causes a reversible, non-selective opening of NPCs. (A) Left panels show influx of fluorescent MBP into nuclei without further addition after 24 and 48 s. Middle panels: simultaneous addition of trans-cyclohexane-1,2-diol (7% w/v) along with the substrate resulted in a greatly enhanced MBP influx. Right panels: nuclei were pre-incubated for 1 min with cyclohexanediol and the reagent was washed out before the influx of MBP was measured. The removal of cyclohexanediol restored the permeability barrier. (B) Comparison of NPC binding of a fluorescent Impβ 45–462 fragment either for untreated nuclei (left) or for nuclei after a 1 min cyclohexanediol treatment and subsequent removal of the reagent. (C) Nuclei were transiently treated with cyclohexanediol as in (A) and (B) and import of the IBB–2×MBP–M3 fusion was measured. The import was strictly receptor dependent, indicating that NPCs had not been irreversibly damaged by the treatment.

Fig. 7. The permeability barrier of NPCs is maintained by weak hydrophobic interactions. The permeability of NPCs for inert molecules was measured as the influx of fluorescent MBP into nuclei. Where indicated, 5% hexane-1,2-diol or hexane-1,2,3-triol was added together with the substrate. Hexanediol, but not the less hydrophobic hexanetriol, caused a non-selective opening of the NPCs and allowed rapid MPB influx. This effect could be fully suppressed by pre-incubation of NPCs with 0.2 mg/ml WGA. For details, see text.