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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

Additional fluctuation traces.(A) RanQ69L•GTP can dissolve existing aggregates. To determine whether RanGTP can dissolve aggregates that have already formed and not just prevent aggregate formation, impβ-YFP•Nup153 aggregates were allowed to form for 5 min at room temperature and their fluorescence fluctuations were measured (blue traces). RanQ69L•GTP was then added to the reaction for 30 min and the fluctuations were measured again (green traces). The disappearance of the majority of large fluorescent spikes indicate that RanQ69L•GTP can dissolve existing aggregates. (B) Test of Ran-reversibility of aggregates formed by an impβ truncation unable to bind Ran. To test the hypothesis that RanQ69L•GTP prevents aggregate formation by displacing impβ that is bound to Nup153FG, we characterized the aggregation capacity of an impβ truncation missing the first 70 N-terminal amino acids, impβ(ΔN70), corresponding to impβ's Ran-binding domain. Impβ(ΔN70)-YFP did not form aggregates on its own (blue trace) but did so when mixed with Nup153FG (green trace). Interestingly, RanQ69L•GTP was now unable to abolish aggregate formation (red trace), indicating that RanGTP dissolves the aggregates through impβ binding and not Nup153FG binding.DOI:http://dx.doi.org/10.7554/eLife.04052.020
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fig6s1: Additional fluctuation traces.(A) RanQ69L•GTP can dissolve existing aggregates. To determine whether RanGTP can dissolve aggregates that have already formed and not just prevent aggregate formation, impβ-YFP•Nup153 aggregates were allowed to form for 5 min at room temperature and their fluorescence fluctuations were measured (blue traces). RanQ69L•GTP was then added to the reaction for 30 min and the fluctuations were measured again (green traces). The disappearance of the majority of large fluorescent spikes indicate that RanQ69L•GTP can dissolve existing aggregates. (B) Test of Ran-reversibility of aggregates formed by an impβ truncation unable to bind Ran. To test the hypothesis that RanQ69L•GTP prevents aggregate formation by displacing impβ that is bound to Nup153FG, we characterized the aggregation capacity of an impβ truncation missing the first 70 N-terminal amino acids, impβ(ΔN70), corresponding to impβ's Ran-binding domain. Impβ(ΔN70)-YFP did not form aggregates on its own (blue trace) but did so when mixed with Nup153FG (green trace). Interestingly, RanQ69L•GTP was now unable to abolish aggregate formation (red trace), indicating that RanGTP dissolves the aggregates through impβ binding and not Nup153FG binding.DOI:http://dx.doi.org/10.7554/eLife.04052.020

Mentions: To explore the notion that impβ and Nup153 act together to form a Ran-sensitive permeability barrier, we investigated their interaction in vitro. Upon co-incubation of recombinant impβ and Nup153FG (the FG domain of Nup153 comprising amino acids 874–1475 [Lim et al., 2006]), large, micron-sized structures formed on a timescale of minutes (Figure 6A). We turned to fluorescence fluctuation spectroscopy (Chen et al., 2000; Tetin, 2013) to examine the structure's assembly and disassembly behaviors and requirements. The fluorescence intensity signal of diffusing impβ-YFP molecules (50 nM) showed a fluctuation pattern characteristic of freely diffusing proteins (Figure 6B). However, when Nup153FG (0.5 µM) was added, large intensity bursts appeared within tens of seconds. The appearance of these spikes in intensity (along with their corresponding long tails in the photon counting histograms) indicated the formation of large impβ•Nup153 complexes (Figure 6B,C, red traces). These higher-order complexes were orders of magnitude brighter than the freely diffusing impβ-YFP, suggesting that they are comprised of tens or even hundreds of impβ molecules. The formation of large complexes can be explained by the many FG motifs found in Nup153's FG domain as well as the multiple sites on impβ's surface that may bind FG repeats. Notably, the addition of RanQ69L•GTP (2 µM), which does not hydrolyze GTP (Bischoff et al., 1994) and is therefore stably in the GTP-bound form, entirely inhibited formation of the complexes. RanQ69L•GTP even dissolved existing large impβ•Nup153FG complexes (Figure 6—figure supplement 1A). This Ran action occurred specifically through impβ (and not Nup153FG) binding since Nup153FG in complex with an impβ truncation lacking the Ran-binding domain, impβ(ΔN70), became insensitive to RanQ69L•GTP (Figure 6—figure supplement 1B).10.7554/eLife.04052.019Figure 6.In vitro formation of large RanGTP-reversible impβ•Nup153FG complexes.


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)

Additional fluctuation traces.(A) RanQ69L•GTP can dissolve existing aggregates. To determine whether RanGTP can dissolve aggregates that have already formed and not just prevent aggregate formation, impβ-YFP•Nup153 aggregates were allowed to form for 5 min at room temperature and their fluorescence fluctuations were measured (blue traces). RanQ69L•GTP was then added to the reaction for 30 min and the fluctuations were measured again (green traces). The disappearance of the majority of large fluorescent spikes indicate that RanQ69L•GTP can dissolve existing aggregates. (B) Test of Ran-reversibility of aggregates formed by an impβ truncation unable to bind Ran. To test the hypothesis that RanQ69L•GTP prevents aggregate formation by displacing impβ that is bound to Nup153FG, we characterized the aggregation capacity of an impβ truncation missing the first 70 N-terminal amino acids, impβ(ΔN70), corresponding to impβ's Ran-binding domain. Impβ(ΔN70)-YFP did not form aggregates on its own (blue trace) but did so when mixed with Nup153FG (green trace). Interestingly, RanQ69L•GTP was now unable to abolish aggregate formation (red trace), indicating that RanGTP dissolves the aggregates through impβ binding and not Nup153FG binding.DOI:http://dx.doi.org/10.7554/eLife.04052.020
© Copyright Policy
Related In: Results  -  Collection

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

fig6s1: Additional fluctuation traces.(A) RanQ69L•GTP can dissolve existing aggregates. To determine whether RanGTP can dissolve aggregates that have already formed and not just prevent aggregate formation, impβ-YFP•Nup153 aggregates were allowed to form for 5 min at room temperature and their fluorescence fluctuations were measured (blue traces). RanQ69L•GTP was then added to the reaction for 30 min and the fluctuations were measured again (green traces). The disappearance of the majority of large fluorescent spikes indicate that RanQ69L•GTP can dissolve existing aggregates. (B) Test of Ran-reversibility of aggregates formed by an impβ truncation unable to bind Ran. To test the hypothesis that RanQ69L•GTP prevents aggregate formation by displacing impβ that is bound to Nup153FG, we characterized the aggregation capacity of an impβ truncation missing the first 70 N-terminal amino acids, impβ(ΔN70), corresponding to impβ's Ran-binding domain. Impβ(ΔN70)-YFP did not form aggregates on its own (blue trace) but did so when mixed with Nup153FG (green trace). Interestingly, RanQ69L•GTP was now unable to abolish aggregate formation (red trace), indicating that RanGTP dissolves the aggregates through impβ binding and not Nup153FG binding.DOI:http://dx.doi.org/10.7554/eLife.04052.020
Mentions: To explore the notion that impβ and Nup153 act together to form a Ran-sensitive permeability barrier, we investigated their interaction in vitro. Upon co-incubation of recombinant impβ and Nup153FG (the FG domain of Nup153 comprising amino acids 874–1475 [Lim et al., 2006]), large, micron-sized structures formed on a timescale of minutes (Figure 6A). We turned to fluorescence fluctuation spectroscopy (Chen et al., 2000; Tetin, 2013) to examine the structure's assembly and disassembly behaviors and requirements. The fluorescence intensity signal of diffusing impβ-YFP molecules (50 nM) showed a fluctuation pattern characteristic of freely diffusing proteins (Figure 6B). However, when Nup153FG (0.5 µM) was added, large intensity bursts appeared within tens of seconds. The appearance of these spikes in intensity (along with their corresponding long tails in the photon counting histograms) indicated the formation of large impβ•Nup153 complexes (Figure 6B,C, red traces). These higher-order complexes were orders of magnitude brighter than the freely diffusing impβ-YFP, suggesting that they are comprised of tens or even hundreds of impβ molecules. The formation of large complexes can be explained by the many FG motifs found in Nup153's FG domain as well as the multiple sites on impβ's surface that may bind FG repeats. Notably, the addition of RanQ69L•GTP (2 µM), which does not hydrolyze GTP (Bischoff et al., 1994) and is therefore stably in the GTP-bound form, entirely inhibited formation of the complexes. RanQ69L•GTP even dissolved existing large impβ•Nup153FG complexes (Figure 6—figure supplement 1A). This Ran action occurred specifically through impβ (and not Nup153FG) binding since Nup153FG in complex with an impβ truncation lacking the Ran-binding domain, impβ(ΔN70), became insensitive to RanQ69L•GTP (Figure 6—figure supplement 1B).10.7554/eLife.04052.019Figure 6.In vitro formation of large RanGTP-reversible impβ•Nup153FG complexes.

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