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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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Strong NESs of Nmd3 on Rio2 bypass requirement for Slx9 but not Yrb2 in 40S pre-ribosome export.(A) rio2-nmd3NES is not synthetic lethal with mex67∆loop or mtr2∆loop116-137. Strains were spotted in 10-fold serial dilutions on 5-FOA (SD) plates and grown at 30°C for 2–4 days (B) The Nmd3-NES (amino acids 440–518) fused to Rio2∆NES bypasses the requirement of the Rio2-NES in export complex formation in vitro. GST-Rio2Nmd3NES was immobilized on GSH-Sepharose and complex formation was carried out and analyzed as in Figure 2C. L = input. (C) rio2-nmd3NES rescues the impaired pre40S export of slx9∆ cells. Localization of uS5-GFP in the indicated strains was monitored by fluorescence microscopy. Scale bar = 5 µm. (D) The rescue of impaired pre40S ribosome export by rio2-nmd3NES is specific for slx9∆. yrb2∆ cells transformed with the indicated plasmids was monitored by fluorescence microscopy for the localization of uS5-GFP. Scale bar = 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05745.012
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fig8: Strong NESs of Nmd3 on Rio2 bypass requirement for Slx9 but not Yrb2 in 40S pre-ribosome export.(A) rio2-nmd3NES is not synthetic lethal with mex67∆loop or mtr2∆loop116-137. Strains were spotted in 10-fold serial dilutions on 5-FOA (SD) plates and grown at 30°C for 2–4 days (B) The Nmd3-NES (amino acids 440–518) fused to Rio2∆NES bypasses the requirement of the Rio2-NES in export complex formation in vitro. GST-Rio2Nmd3NES was immobilized on GSH-Sepharose and complex formation was carried out and analyzed as in Figure 2C. L = input. (C) rio2-nmd3NES rescues the impaired pre40S export of slx9∆ cells. Localization of uS5-GFP in the indicated strains was monitored by fluorescence microscopy. Scale bar = 5 µm. (D) The rescue of impaired pre40S ribosome export by rio2-nmd3NES is specific for slx9∆. yrb2∆ cells transformed with the indicated plasmids was monitored by fluorescence microscopy for the localization of uS5-GFP. Scale bar = 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05745.012

Mentions: Functional analyses revealed that the expression of Rio2Nmd3NES complemented the lethality of the rio2∆ strain. Moreover, rio2-nmd3NES was not synthetic lethal with mex67∆loop and mtr2∆116-137 (Figure 8A). Since Rio2Nmd3NES bound to Crm1 in the presence of RanGTP (Figure 8B, lane 4), we assessed whether Rio2Nmd3NES expression rescued the 40S pre-ribosome export defect seen in slx9∆ cells. >95% of slx9∆ cells expressing Rio2 and Rio2∆NES accumulated uS5-GFP in the nucleoplasm. However, slx9∆ cells expressing Rio2Nmd3NES did not accumulate uS5-GFP in the nucleoplasm (Figure 8C), indicating no apparent impairment in 40S pre-ribosome export. A Rio2 variant containing only the first NES of Nmd3 (rio2-nmd3NES∆1) was unable to rescue the 40S pre-ribosome export defect of slx9∆ cells (Figure 8C), suggesting that both NESs are required to bypass Slx9 function in 40S pre-ribosome export. Notably, the expression of Rio2Nmd3NES in yrb2∆ cells did not rescue the nucleoplasmic accumulation of uS5-GFP (Figure 8D), indicating that the heterologous NESs specifically bypasses Slx9 function but not other steps that drive 40S pre-ribosome export.10.7554/eLife.05745.012Figure 8.Strong NESs of Nmd3 on Rio2 bypass requirement for Slx9 but not Yrb2 in 40S pre-ribosome export.


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)

Strong NESs of Nmd3 on Rio2 bypass requirement for Slx9 but not Yrb2 in 40S pre-ribosome export.(A) rio2-nmd3NES is not synthetic lethal with mex67∆loop or mtr2∆loop116-137. Strains were spotted in 10-fold serial dilutions on 5-FOA (SD) plates and grown at 30°C for 2–4 days (B) The Nmd3-NES (amino acids 440–518) fused to Rio2∆NES bypasses the requirement of the Rio2-NES in export complex formation in vitro. GST-Rio2Nmd3NES was immobilized on GSH-Sepharose and complex formation was carried out and analyzed as in Figure 2C. L = input. (C) rio2-nmd3NES rescues the impaired pre40S export of slx9∆ cells. Localization of uS5-GFP in the indicated strains was monitored by fluorescence microscopy. Scale bar = 5 µm. (D) The rescue of impaired pre40S ribosome export by rio2-nmd3NES is specific for slx9∆. yrb2∆ cells transformed with the indicated plasmids was monitored by fluorescence microscopy for the localization of uS5-GFP. Scale bar = 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05745.012
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4402694&req=5

fig8: Strong NESs of Nmd3 on Rio2 bypass requirement for Slx9 but not Yrb2 in 40S pre-ribosome export.(A) rio2-nmd3NES is not synthetic lethal with mex67∆loop or mtr2∆loop116-137. Strains were spotted in 10-fold serial dilutions on 5-FOA (SD) plates and grown at 30°C for 2–4 days (B) The Nmd3-NES (amino acids 440–518) fused to Rio2∆NES bypasses the requirement of the Rio2-NES in export complex formation in vitro. GST-Rio2Nmd3NES was immobilized on GSH-Sepharose and complex formation was carried out and analyzed as in Figure 2C. L = input. (C) rio2-nmd3NES rescues the impaired pre40S export of slx9∆ cells. Localization of uS5-GFP in the indicated strains was monitored by fluorescence microscopy. Scale bar = 5 µm. (D) The rescue of impaired pre40S ribosome export by rio2-nmd3NES is specific for slx9∆. yrb2∆ cells transformed with the indicated plasmids was monitored by fluorescence microscopy for the localization of uS5-GFP. Scale bar = 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05745.012
Mentions: Functional analyses revealed that the expression of Rio2Nmd3NES complemented the lethality of the rio2∆ strain. Moreover, rio2-nmd3NES was not synthetic lethal with mex67∆loop and mtr2∆116-137 (Figure 8A). Since Rio2Nmd3NES bound to Crm1 in the presence of RanGTP (Figure 8B, lane 4), we assessed whether Rio2Nmd3NES expression rescued the 40S pre-ribosome export defect seen in slx9∆ cells. >95% of slx9∆ cells expressing Rio2 and Rio2∆NES accumulated uS5-GFP in the nucleoplasm. However, slx9∆ cells expressing Rio2Nmd3NES did not accumulate uS5-GFP in the nucleoplasm (Figure 8C), indicating no apparent impairment in 40S pre-ribosome export. A Rio2 variant containing only the first NES of Nmd3 (rio2-nmd3NES∆1) was unable to rescue the 40S pre-ribosome export defect of slx9∆ cells (Figure 8C), suggesting that both NESs are required to bypass Slx9 function in 40S pre-ribosome export. Notably, the expression of Rio2Nmd3NES in yrb2∆ cells did not rescue the nucleoplasmic accumulation of uS5-GFP (Figure 8D), indicating that the heterologous NESs specifically bypasses Slx9 function but not other steps that drive 40S pre-ribosome export.10.7554/eLife.05745.012Figure 8.Strong NESs of Nmd3 on Rio2 bypass requirement for Slx9 but not Yrb2 in 40S pre-ribosome export.

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
Related in: MedlinePlus