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A non-canonical mechanism for Crm1-export cargo complex assembly.

Fischer U, Schäuble N, Schütz S, Altvater M, Chang Y, Faza MB, Panse VG - Elife (2015)

Bottom Line: In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2.Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export.This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland.

ABSTRACT
The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.

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The basic patch and acidic tail of Ran modulates interactions with Slx9.(A) The basic patch of RanQLGTP contributes to Slx9 binding. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP, His6-RanQLGTPRKAA, or His6-RanQLGTPRKEE). After washing, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibody. L = input. (B) The acidic tail of RanQLGTP negatively regulates interactions with Slx9. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP or His6-Ran∆CQLGTP). Analysis of the eluted proteins was carried out as described in (A). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.005
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fig3: The basic patch and acidic tail of Ran modulates interactions with Slx9.(A) The basic patch of RanQLGTP contributes to Slx9 binding. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP, His6-RanQLGTPRKAA, or His6-RanQLGTPRKEE). After washing, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibody. L = input. (B) The acidic tail of RanQLGTP negatively regulates interactions with Slx9. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP or His6-Ran∆CQLGTP). Analysis of the eluted proteins was carried out as described in (A). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.005

Mentions: A conserved basic patch on Ran is involved in the interaction with known Ran-binding proteins (Nilsson et al., 2001). Based on homology to human Ran, arginine 142, and lysine 143 in yeast Ran were mutated to alanine (RanQLGTPRKAA) or glutamate (RanQLGTPRKEE) and the contribution of this basic patch to Slx9:RanGTP complex formation was analyzed in vitro. In agreement with previous studies (Nilsson et al., 2001), these RanQLGTP mutants bound weakly to the importin β-like transport receptor, Kap123 (Figure 3A, compare lane 10 with lanes 11 and 12), and interacted more strongly with the RanBP1 homolog Yrb1 (Figure 3A, compare lane 6 with lanes 7 and 8). Pull down studies of Slx9 and these Ran mutants showed that the interactions between Slx9 and these two Ran mutants were impaired, with the charge reversal mutant having a more severe effect than the alanine mutant (Figure 3A, compare lane 2 with lanes 3 and 4). Altogether, these results suggest that, similar to Kap123, Slx9 binding to RanQLGTP involves the basic patch.10.7554/eLife.05745.005Figure 3.The basic patch and acidic tail of Ran modulates interactions with Slx9.


A non-canonical mechanism for Crm1-export cargo complex assembly.

Fischer U, Schäuble N, Schütz S, Altvater M, Chang Y, Faza MB, Panse VG - Elife (2015)

The basic patch and acidic tail of Ran modulates interactions with Slx9.(A) The basic patch of RanQLGTP contributes to Slx9 binding. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP, His6-RanQLGTPRKAA, or His6-RanQLGTPRKEE). After washing, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibody. L = input. (B) The acidic tail of RanQLGTP negatively regulates interactions with Slx9. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP or His6-Ran∆CQLGTP). Analysis of the eluted proteins was carried out as described in (A). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.005
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Related In: Results  -  Collection

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fig3: The basic patch and acidic tail of Ran modulates interactions with Slx9.(A) The basic patch of RanQLGTP contributes to Slx9 binding. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP, His6-RanQLGTPRKAA, or His6-RanQLGTPRKEE). After washing, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibody. L = input. (B) The acidic tail of RanQLGTP negatively regulates interactions with Slx9. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP or His6-Ran∆CQLGTP). Analysis of the eluted proteins was carried out as described in (A). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.005
Mentions: A conserved basic patch on Ran is involved in the interaction with known Ran-binding proteins (Nilsson et al., 2001). Based on homology to human Ran, arginine 142, and lysine 143 in yeast Ran were mutated to alanine (RanQLGTPRKAA) or glutamate (RanQLGTPRKEE) and the contribution of this basic patch to Slx9:RanGTP complex formation was analyzed in vitro. In agreement with previous studies (Nilsson et al., 2001), these RanQLGTP mutants bound weakly to the importin β-like transport receptor, Kap123 (Figure 3A, compare lane 10 with lanes 11 and 12), and interacted more strongly with the RanBP1 homolog Yrb1 (Figure 3A, compare lane 6 with lanes 7 and 8). Pull down studies of Slx9 and these Ran mutants showed that the interactions between Slx9 and these two Ran mutants were impaired, with the charge reversal mutant having a more severe effect than the alanine mutant (Figure 3A, compare lane 2 with lanes 3 and 4). Altogether, these results suggest that, similar to Kap123, Slx9 binding to RanQLGTP involves the basic patch.10.7554/eLife.05745.005Figure 3.The basic patch and acidic tail of Ran modulates interactions with Slx9.

Bottom Line: In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2.Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export.This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland.

ABSTRACT
The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.

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