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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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Genetic interactions between Rio2 alleles and RanGTP-binding proteins Slx9 and Yrb2.(A) slx9∆ and slx9-1 do not genetically interact with rio2∆NES. A RIO2 shuffle slx9∆ strain was transformed with the indicated combinations of empty, WT, or mutant plasmids and spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days. (B) rio2∆NES weakly genetically interacts with yrb2∆. RIO2 shuffle yrb2∆ strain transformed with the indicated combinations of empty, WT or mutant plasmids were spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days.DOI:http://dx.doi.org/10.7554/eLife.05745.011
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fig7s2: Genetic interactions between Rio2 alleles and RanGTP-binding proteins Slx9 and Yrb2.(A) slx9∆ and slx9-1 do not genetically interact with rio2∆NES. A RIO2 shuffle slx9∆ strain was transformed with the indicated combinations of empty, WT, or mutant plasmids and spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days. (B) rio2∆NES weakly genetically interacts with yrb2∆. RIO2 shuffle yrb2∆ strain transformed with the indicated combinations of empty, WT or mutant plasmids were spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days.DOI:http://dx.doi.org/10.7554/eLife.05745.011

Mentions: We next assessed the functionality of Rio2∆NES and Rio23G in yeast. Both rio2∆NES and rio23G rescued the lethality of the rio2∆ strain (Figure 7B). However, rio2∆NES was synthetically lethal mex67∆loop and mtr2∆116-137 (Figure 7B), consistent with the model that the transport receptor Mex67-Mtr2 has a redundant function in 40S pre-ribosome nuclear export. Curiously, the rio23G allele that did not recruit Crm1 in the presence of RanQLGTP in vitro (Figure 7A, bottom panel, lane 4), rescued the synthetic lethality (Figure 7B), indicating that Rio23G is still functional in vivo. Importantly, neither rio2 alleles (rio2∆NES and rio23G) were synthetic lethal when combined with slx9∆ and slx9-1 mutant strains (Figure 7—figure supplement 2A).


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)

Genetic interactions between Rio2 alleles and RanGTP-binding proteins Slx9 and Yrb2.(A) slx9∆ and slx9-1 do not genetically interact with rio2∆NES. A RIO2 shuffle slx9∆ strain was transformed with the indicated combinations of empty, WT, or mutant plasmids and spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days. (B) rio2∆NES weakly genetically interacts with yrb2∆. RIO2 shuffle yrb2∆ strain transformed with the indicated combinations of empty, WT or mutant plasmids were spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days.DOI:http://dx.doi.org/10.7554/eLife.05745.011
© Copyright Policy
Related In: Results  -  Collection

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

fig7s2: Genetic interactions between Rio2 alleles and RanGTP-binding proteins Slx9 and Yrb2.(A) slx9∆ and slx9-1 do not genetically interact with rio2∆NES. A RIO2 shuffle slx9∆ strain was transformed with the indicated combinations of empty, WT, or mutant plasmids and spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days. (B) rio2∆NES weakly genetically interacts with yrb2∆. RIO2 shuffle yrb2∆ strain transformed with the indicated combinations of empty, WT or mutant plasmids were spotted in 10-fold dilutions on SD-plates containing 5-FOA and grown at 25°C for 2–4 days.DOI:http://dx.doi.org/10.7554/eLife.05745.011
Mentions: We next assessed the functionality of Rio2∆NES and Rio23G in yeast. Both rio2∆NES and rio23G rescued the lethality of the rio2∆ strain (Figure 7B). However, rio2∆NES was synthetically lethal mex67∆loop and mtr2∆116-137 (Figure 7B), consistent with the model that the transport receptor Mex67-Mtr2 has a redundant function in 40S pre-ribosome nuclear export. Curiously, the rio23G allele that did not recruit Crm1 in the presence of RanQLGTP in vitro (Figure 7A, bottom panel, lane 4), rescued the synthetic lethality (Figure 7B), indicating that Rio23G is still functional in vivo. Importantly, neither rio2 alleles (rio2∆NES and rio23G) were synthetic lethal when combined with slx9∆ and slx9-1 mutant strains (Figure 7—figure supplement 2A).

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