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Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner.

Lowe AR, Tang JH, Yassif J, Graf M, Huang WY, Groves JT, Weis K, Liphardt JT - Elife (2015)

Bottom Line: A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153.Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired.RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153.

View Article: PubMed Central - PubMed

Affiliation: Institute for Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom.

ABSTRACT
Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.

No MeSH data available.


Related in: MedlinePlus

RanGTP and a Ran ‘wash’ increase the passive equilibration of GFP2 into wild type nuclei even when no impβ is present.In the GFP2 passive equilibration assays, it was intriguing that RanGTP made the pore more permeable than when impβ was excluded. We hypothesized that perhaps there was a significant amount of endogenous impβ, as well as other transport receptors, still residing at the pore that were not removed even after several wash steps during the digitonin permeabilization procedure. To test this, we performed an extra RanGTP wash step, in which Ran, GTP, and an energy-regeneration system were incubated with the nuclei for 10 min before being washed away. During this step, RanGTP will bind to and dissociate any endogenous transport receptors still bound to the NPC which are then subsequently washed away. In comparison to the condition in which impβ and RanGTP are not added to the nuclei, the RanGTP wash did indeed make the NPCs more permeable (red curve), although they were still not as permeable as when impβ and RanGTP are added together or when RanGTP is added alone (green and purple curves). This trend suggests that a subpopulation of endogenous transport receptors still reside at the NPC even after digitonin permeabilization.DOI:http://dx.doi.org/10.7554/eLife.04052.018
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fig5s1: RanGTP and a Ran ‘wash’ increase the passive equilibration of GFP2 into wild type nuclei even when no impβ is present.In the GFP2 passive equilibration assays, it was intriguing that RanGTP made the pore more permeable than when impβ was excluded. We hypothesized that perhaps there was a significant amount of endogenous impβ, as well as other transport receptors, still residing at the pore that were not removed even after several wash steps during the digitonin permeabilization procedure. To test this, we performed an extra RanGTP wash step, in which Ran, GTP, and an energy-regeneration system were incubated with the nuclei for 10 min before being washed away. During this step, RanGTP will bind to and dissociate any endogenous transport receptors still bound to the NPC which are then subsequently washed away. In comparison to the condition in which impβ and RanGTP are not added to the nuclei, the RanGTP wash did indeed make the NPCs more permeable (red curve), although they were still not as permeable as when impβ and RanGTP are added together or when RanGTP is added alone (green and purple curves). This trend suggests that a subpopulation of endogenous transport receptors still reside at the NPC even after digitonin permeabilization.DOI:http://dx.doi.org/10.7554/eLife.04052.018

Mentions: We found that 1 μM impβ significantly decreased the permeability of the NPC for the GFP1 and GFP2 probes (Figure 5C,D). By contrast, when RanGTP was also added, the permeability was greatly increased. The GFP3 probe translocated across the NPC at a relatively slow rate with or without impβ and RanGTP, likely because GFP3's size is considerably larger than the passive diffusion size cutoff of the pore (Figure 5C). We therefore decided to focus on the GFP2 probe and we used it to further explore the effects of impβ and Nup153 on the permeability of the NPC (Figure 5D,E). First, we tested the possibility that general molecular crowding, for instance due to widespread transport receptor-FG nucleoporin interactions, was responsible for the observed permeability modulation by impβ. We therefore repeated the previous experiments with the related transport receptor transportin-1 (TRN1). TRN1 is the transport receptor for M9 signal peptide-containing cargos such as hnRNPs and belongs to the same class of karyopherins as impβ (Pollard et al., 1996). At 1 and even 2 μM TRN1, there was no strong effect on NPC permeability (Figure 5E), suggesting that general molecular crowding is not responsible for the changes to NPC permeability. Importantly, for Δ15370% nuclei, addition of impβ no longer restricted the NPC (Figure 5D). Together, these results suggest that specifically the impβ•Nup153 interaction causes the nuclear pore to become less permeable. Furthermore, because the inert probes undergo purely passive translocation across the NPC, the reduced permeability must be due to a specific steric ‘barrier’ within the pore and not due to a block of transport receptor-specific binding sites. This steric barrier appears to involve impβ•Nup153 interactions that are very stable and long-lived in the absence of RanGTP. Indeed RanGTP causes the pore to become more permeable even when no exogenous impβ is first added. This is likely due to endogenous impβ and other transport receptors residing in the pore that were not washed away during digitonin permeabilization (Figure 5—figure supplement 1).


Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner.

Lowe AR, Tang JH, Yassif J, Graf M, Huang WY, Groves JT, Weis K, Liphardt JT - Elife (2015)

RanGTP and a Ran ‘wash’ increase the passive equilibration of GFP2 into wild type nuclei even when no impβ is present.In the GFP2 passive equilibration assays, it was intriguing that RanGTP made the pore more permeable than when impβ was excluded. We hypothesized that perhaps there was a significant amount of endogenous impβ, as well as other transport receptors, still residing at the pore that were not removed even after several wash steps during the digitonin permeabilization procedure. To test this, we performed an extra RanGTP wash step, in which Ran, GTP, and an energy-regeneration system were incubated with the nuclei for 10 min before being washed away. During this step, RanGTP will bind to and dissociate any endogenous transport receptors still bound to the NPC which are then subsequently washed away. In comparison to the condition in which impβ and RanGTP are not added to the nuclei, the RanGTP wash did indeed make the NPCs more permeable (red curve), although they were still not as permeable as when impβ and RanGTP are added together or when RanGTP is added alone (green and purple curves). This trend suggests that a subpopulation of endogenous transport receptors still reside at the NPC even after digitonin permeabilization.DOI:http://dx.doi.org/10.7554/eLife.04052.018
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4375889&req=5

fig5s1: RanGTP and a Ran ‘wash’ increase the passive equilibration of GFP2 into wild type nuclei even when no impβ is present.In the GFP2 passive equilibration assays, it was intriguing that RanGTP made the pore more permeable than when impβ was excluded. We hypothesized that perhaps there was a significant amount of endogenous impβ, as well as other transport receptors, still residing at the pore that were not removed even after several wash steps during the digitonin permeabilization procedure. To test this, we performed an extra RanGTP wash step, in which Ran, GTP, and an energy-regeneration system were incubated with the nuclei for 10 min before being washed away. During this step, RanGTP will bind to and dissociate any endogenous transport receptors still bound to the NPC which are then subsequently washed away. In comparison to the condition in which impβ and RanGTP are not added to the nuclei, the RanGTP wash did indeed make the NPCs more permeable (red curve), although they were still not as permeable as when impβ and RanGTP are added together or when RanGTP is added alone (green and purple curves). This trend suggests that a subpopulation of endogenous transport receptors still reside at the NPC even after digitonin permeabilization.DOI:http://dx.doi.org/10.7554/eLife.04052.018
Mentions: We found that 1 μM impβ significantly decreased the permeability of the NPC for the GFP1 and GFP2 probes (Figure 5C,D). By contrast, when RanGTP was also added, the permeability was greatly increased. The GFP3 probe translocated across the NPC at a relatively slow rate with or without impβ and RanGTP, likely because GFP3's size is considerably larger than the passive diffusion size cutoff of the pore (Figure 5C). We therefore decided to focus on the GFP2 probe and we used it to further explore the effects of impβ and Nup153 on the permeability of the NPC (Figure 5D,E). First, we tested the possibility that general molecular crowding, for instance due to widespread transport receptor-FG nucleoporin interactions, was responsible for the observed permeability modulation by impβ. We therefore repeated the previous experiments with the related transport receptor transportin-1 (TRN1). TRN1 is the transport receptor for M9 signal peptide-containing cargos such as hnRNPs and belongs to the same class of karyopherins as impβ (Pollard et al., 1996). At 1 and even 2 μM TRN1, there was no strong effect on NPC permeability (Figure 5E), suggesting that general molecular crowding is not responsible for the changes to NPC permeability. Importantly, for Δ15370% nuclei, addition of impβ no longer restricted the NPC (Figure 5D). Together, these results suggest that specifically the impβ•Nup153 interaction causes the nuclear pore to become less permeable. Furthermore, because the inert probes undergo purely passive translocation across the NPC, the reduced permeability must be due to a specific steric ‘barrier’ within the pore and not due to a block of transport receptor-specific binding sites. This steric barrier appears to involve impβ•Nup153 interactions that are very stable and long-lived in the absence of RanGTP. Indeed RanGTP causes the pore to become more permeable even when no exogenous impβ is first added. This is likely due to endogenous impβ and other transport receptors residing in the pore that were not washed away during digitonin permeabilization (Figure 5—figure supplement 1).

Bottom Line: A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153.Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired.RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153.

View Article: PubMed Central - PubMed

Affiliation: Institute for Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom.

ABSTRACT
Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.

No MeSH data available.


Related in: MedlinePlus