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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 ‘flexibility’ of the NES region in Rio2 contributes to its interaction with Crm1 in the presence of RanGTP.Top: schematic of Rio2 highlighting the triple A mutation (399-EEN-401-AAA, brown) proximal to the NES. Hydrophobic amino acids of the NES are red and mutated amino acids are brown. Bottom: immobilized GST-Rio23A was incubated with buffer alone or buffer containing 2 µM His6-RanQLGTP, 50 nM Crm1-His6 or 2 µM His6-RanQLGTP, and 50 nM Crm1-His6. After washing, eluted proteins were separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using indicated antibodies. L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.010
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fig7s1: The ‘flexibility’ of the NES region in Rio2 contributes to its interaction with Crm1 in the presence of RanGTP.Top: schematic of Rio2 highlighting the triple A mutation (399-EEN-401-AAA, brown) proximal to the NES. Hydrophobic amino acids of the NES are red and mutated amino acids are brown. Bottom: immobilized GST-Rio23A was incubated with buffer alone or buffer containing 2 µM His6-RanQLGTP, 50 nM Crm1-His6 or 2 µM His6-RanQLGTP, and 50 nM Crm1-His6. After washing, eluted proteins were separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using indicated antibodies. L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.010

Mentions: Crm1 recognizes and binds cargos that contain diverse leucine-rich NESs. Structural analyses of the RanGTP:Crm1 complex bound to prototypic NESs suggest that any peptide can function as a NES as long as its backbone conformation permits its side chains to access the rigid hydrophobic pockets of Crm1 (Güttler et al., 2010). To test whether conformational rigidity of the Rio2-NES is critical to recruit Crm1 in the presence of RanQLGTP, three consecutive residues (399-EEN-401) proximal to the NES were mutated to glycines (Rio23G) (Figure 7A, top panel). Because glycine residues lack a side chain, they allow greater conformational flexibility for the polypeptide backbone of these residues (Ramachandran and Sasisekharan, 1968) as well as to the neighboring NES, thus destabilizing it. We found that, like the Rio2∆NES (Figure 4A, lane 8), Rio23G was unable to cooperatively recruit Crm1 in the presence of RanQLGTP in vitro (Figure 7A, bottom panel, lane 4). In parallel, we made a Rio2 mutant in which residues 399–401 were replaced by alanines (Rio23A) (Figure 7—figure supplement 1, top panel). Unlike Rio23G (Figure 7A, bottom panel, lane 4), Rio23A was able to efficiently cooperatively recruit Crm1 in the presence of RanQLGTP (Figure 7—figure supplement 1, bottom panel), suggesting that the glycine mutations destabilize the NES.10.7554/eLife.05745.009Figure 7.Slx9 provides a scaffold to load Crm1 onto Rio2-NES.


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 ‘flexibility’ of the NES region in Rio2 contributes to its interaction with Crm1 in the presence of RanGTP.Top: schematic of Rio2 highlighting the triple A mutation (399-EEN-401-AAA, brown) proximal to the NES. Hydrophobic amino acids of the NES are red and mutated amino acids are brown. Bottom: immobilized GST-Rio23A was incubated with buffer alone or buffer containing 2 µM His6-RanQLGTP, 50 nM Crm1-His6 or 2 µM His6-RanQLGTP, and 50 nM Crm1-His6. After washing, eluted proteins were separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using indicated antibodies. L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.010
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402694&req=5

fig7s1: The ‘flexibility’ of the NES region in Rio2 contributes to its interaction with Crm1 in the presence of RanGTP.Top: schematic of Rio2 highlighting the triple A mutation (399-EEN-401-AAA, brown) proximal to the NES. Hydrophobic amino acids of the NES are red and mutated amino acids are brown. Bottom: immobilized GST-Rio23A was incubated with buffer alone or buffer containing 2 µM His6-RanQLGTP, 50 nM Crm1-His6 or 2 µM His6-RanQLGTP, and 50 nM Crm1-His6. After washing, eluted proteins were separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using indicated antibodies. L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.010
Mentions: Crm1 recognizes and binds cargos that contain diverse leucine-rich NESs. Structural analyses of the RanGTP:Crm1 complex bound to prototypic NESs suggest that any peptide can function as a NES as long as its backbone conformation permits its side chains to access the rigid hydrophobic pockets of Crm1 (Güttler et al., 2010). To test whether conformational rigidity of the Rio2-NES is critical to recruit Crm1 in the presence of RanQLGTP, three consecutive residues (399-EEN-401) proximal to the NES were mutated to glycines (Rio23G) (Figure 7A, top panel). Because glycine residues lack a side chain, they allow greater conformational flexibility for the polypeptide backbone of these residues (Ramachandran and Sasisekharan, 1968) as well as to the neighboring NES, thus destabilizing it. We found that, like the Rio2∆NES (Figure 4A, lane 8), Rio23G was unable to cooperatively recruit Crm1 in the presence of RanQLGTP in vitro (Figure 7A, bottom panel, lane 4). In parallel, we made a Rio2 mutant in which residues 399–401 were replaced by alanines (Rio23A) (Figure 7—figure supplement 1, top panel). Unlike Rio23G (Figure 7A, bottom panel, lane 4), Rio23A was able to efficiently cooperatively recruit Crm1 in the presence of RanQLGTP (Figure 7—figure supplement 1, bottom panel), suggesting that the glycine mutations destabilize the NES.10.7554/eLife.05745.009Figure 7.Slx9 provides a scaffold to load Crm1 onto Rio2-NES.

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.

Show MeSH