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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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Comparison of Coomassie staining and Western blot signals of Slx9 and Slx9-1 proteins. 0.5 µM Slx9 or 0.5 µM Slx9-1 were separated on SDS-PAGE (top panel) and either stained with Coomassie or detected by Western analysis with Slx9 antibody (lower panel). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.017
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fig9: Comparison of Coomassie staining and Western blot signals of Slx9 and Slx9-1 proteins. 0.5 µM Slx9 or 0.5 µM Slx9-1 were separated on SDS-PAGE (top panel) and either stained with Coomassie or detected by Western analysis with Slx9 antibody (lower panel). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.017

Mentions: We agree with the reviewers that there is a slightly stronger signal for Slx9-1 in the Western blot. We have analyzed the differences by carefully running equalized samples of the two proteins, and this difference is small, but present (Author response image 1). However, whether Slx9-1 binds slightly better to GST-Rio2 in vitro or to the pre-ribosomal particle (Enp1-TAP) in vivo (Figure 1C) does not change the major conclusion of our experiments: Slx9-1 is expressed in vivo and recruited to the 40S pre-ribosome. Therefore, we have altered the description of the interaction studies to say that Slx9-1 binds at least as well as the wild-type.10.7554/eLife.05745.017Author response image 1.


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)

Comparison of Coomassie staining and Western blot signals of Slx9 and Slx9-1 proteins. 0.5 µM Slx9 or 0.5 µM Slx9-1 were separated on SDS-PAGE (top panel) and either stained with Coomassie or detected by Western analysis with Slx9 antibody (lower panel). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.017
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Comparison of Coomassie staining and Western blot signals of Slx9 and Slx9-1 proteins. 0.5 µM Slx9 or 0.5 µM Slx9-1 were separated on SDS-PAGE (top panel) and either stained with Coomassie or detected by Western analysis with Slx9 antibody (lower panel). L = input.DOI:http://dx.doi.org/10.7554/eLife.05745.017
Mentions: We agree with the reviewers that there is a slightly stronger signal for Slx9-1 in the Western blot. We have analyzed the differences by carefully running equalized samples of the two proteins, and this difference is small, but present (Author response image 1). However, whether Slx9-1 binds slightly better to GST-Rio2 in vitro or to the pre-ribosomal particle (Enp1-TAP) in vivo (Figure 1C) does not change the major conclusion of our experiments: Slx9-1 is expressed in vivo and recruited to the 40S pre-ribosome. Therefore, we have altered the description of the interaction studies to say that Slx9-1 binds at least as well as the wild-type.10.7554/eLife.05745.017Author response image 1.

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