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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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Slx9 is a RanGTP binding protein.(A) slx9-1 genetically interacts with factors involved in 40S pre-ribosome export. slx9-1 is synthetically lethal with mex67∆loop, mtr2∆loop116-137, or yrb2∆ and strongly synthetically enhanced with rrp12-GFP. Strains containing the indicated WT and mutant alleles were spotted in 10-fold serial dilutions on 5-FOA-SD or SD and grown at 20–30°C for 3–6 days. (B) Slx9 shuttles between the nucleus and the cytoplasm. Cells expressing Enp1-GFP, Gar1-GFP, or Slx9-GFP were mated with kar1-1 cells expressing Nup82-mCherry. The resulting heterokaryons were analyzed by fluorescence microscopy. Scale bar = 5 µm. (C) Slx9 directly binds to RanGTP. GST-Slx9 or GST-Ssb1C was immobilized on GSH-Sepharose before incubating with either 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, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibodies. L = input. (D) Slx9 specifically interacts with the GTP-bound form of Ran. GST-Slx9, GST-Yrb1, or GST-Ntf2 was immobilized on GSH-Sepharose and incubated with buffer alone or 2 µM His6-Ran loaded with GDP or GTP. Analysis of the eluted proteins was carried out as described in (C). L = input. (E) Slx9-1 binding to RanGTP is impaired. Top: GST-Slx9 or GST-Slx9-1 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM His6-RanQLGTP. Analysis of the eluted proteins was carried out as described in (C). L = input. Bottom: bar graph depicts the bound His6-RanQLGTP Western blot signal normalized to GST-Slx9 and GST-Slx9-1 levels, respectively. Four independent experiments were performed and Western blots were quantified by software ImageJ (Version 1.44o). Error bars (S.D.) are indicated.DOI:http://dx.doi.org/10.7554/eLife.05745.004
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fig2: Slx9 is a RanGTP binding protein.(A) slx9-1 genetically interacts with factors involved in 40S pre-ribosome export. slx9-1 is synthetically lethal with mex67∆loop, mtr2∆loop116-137, or yrb2∆ and strongly synthetically enhanced with rrp12-GFP. Strains containing the indicated WT and mutant alleles were spotted in 10-fold serial dilutions on 5-FOA-SD or SD and grown at 20–30°C for 3–6 days. (B) Slx9 shuttles between the nucleus and the cytoplasm. Cells expressing Enp1-GFP, Gar1-GFP, or Slx9-GFP were mated with kar1-1 cells expressing Nup82-mCherry. The resulting heterokaryons were analyzed by fluorescence microscopy. Scale bar = 5 µm. (C) Slx9 directly binds to RanGTP. GST-Slx9 or GST-Ssb1C was immobilized on GSH-Sepharose before incubating with either 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, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibodies. L = input. (D) Slx9 specifically interacts with the GTP-bound form of Ran. GST-Slx9, GST-Yrb1, or GST-Ntf2 was immobilized on GSH-Sepharose and incubated with buffer alone or 2 µM His6-Ran loaded with GDP or GTP. Analysis of the eluted proteins was carried out as described in (C). L = input. (E) Slx9-1 binding to RanGTP is impaired. Top: GST-Slx9 or GST-Slx9-1 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM His6-RanQLGTP. Analysis of the eluted proteins was carried out as described in (C). L = input. Bottom: bar graph depicts the bound His6-RanQLGTP Western blot signal normalized to GST-Slx9 and GST-Slx9-1 levels, respectively. Four independent experiments were performed and Western blots were quantified by software ImageJ (Version 1.44o). Error bars (S.D.) are indicated.DOI:http://dx.doi.org/10.7554/eLife.05745.004

Mentions: Mutations in MEX67 and MTR2 (mex67Δloop and mtr2Δloop116-137), which encode the essential transport receptor Mex67-Mtr2, are synthetically lethal when combined with the slx9Δ mutant (Faza et al., 2012). In addition, we found that slx9∆ displayed a synthetic growth defect with a strain expressing Rrp12-GFP (Figure 2A). Rrp12 is a 40S pre-ribosome export factor that directly interacts with FG-rich nucleoporins (Oeffinger et al., 2004). Based on these genetic interactions, we asked whether Slx9 functions as a novel export factor for the 40S pre-ribosome. A salient feature of an export factor is that it rapidly shuttles between the nucleus and the cytoplasm. To test this, we employed the established heterokaryon assay (Altvater et al., 2014). WT cells expressing Slx9-GFP were mated to kar1-1 cells, which are deficient in nuclear fusion after cell conjugation, leading to heterokaryon formation. In order to distinguish between the two nuclei, kar1-1 cells also contained Nup82-mCherry as a marker for nuclear pores. As controls, we used the shuttling 40S assembly factor Enp1 and the non-shuttling nucleolar protein Gar1 fused to GFP. Whereas Gar1-GFP was never seen in the nucleus of kar1-1 cells (red signal), both Enp1-GFP and Slx9-GFP localized to both nuclei (Figure 2B). These data are consistent with the shuttling of Slx9 between the nuclear and the cytoplasmic compartments.10.7554/eLife.05745.004Figure 2.Slx9 is a RanGTP binding protein.


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)

Slx9 is a RanGTP binding protein.(A) slx9-1 genetically interacts with factors involved in 40S pre-ribosome export. slx9-1 is synthetically lethal with mex67∆loop, mtr2∆loop116-137, or yrb2∆ and strongly synthetically enhanced with rrp12-GFP. Strains containing the indicated WT and mutant alleles were spotted in 10-fold serial dilutions on 5-FOA-SD or SD and grown at 20–30°C for 3–6 days. (B) Slx9 shuttles between the nucleus and the cytoplasm. Cells expressing Enp1-GFP, Gar1-GFP, or Slx9-GFP were mated with kar1-1 cells expressing Nup82-mCherry. The resulting heterokaryons were analyzed by fluorescence microscopy. Scale bar = 5 µm. (C) Slx9 directly binds to RanGTP. GST-Slx9 or GST-Ssb1C was immobilized on GSH-Sepharose before incubating with either 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, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibodies. L = input. (D) Slx9 specifically interacts with the GTP-bound form of Ran. GST-Slx9, GST-Yrb1, or GST-Ntf2 was immobilized on GSH-Sepharose and incubated with buffer alone or 2 µM His6-Ran loaded with GDP or GTP. Analysis of the eluted proteins was carried out as described in (C). L = input. (E) Slx9-1 binding to RanGTP is impaired. Top: GST-Slx9 or GST-Slx9-1 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM His6-RanQLGTP. Analysis of the eluted proteins was carried out as described in (C). L = input. Bottom: bar graph depicts the bound His6-RanQLGTP Western blot signal normalized to GST-Slx9 and GST-Slx9-1 levels, respectively. Four independent experiments were performed and Western blots were quantified by software ImageJ (Version 1.44o). Error bars (S.D.) are indicated.DOI:http://dx.doi.org/10.7554/eLife.05745.004
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fig2: Slx9 is a RanGTP binding protein.(A) slx9-1 genetically interacts with factors involved in 40S pre-ribosome export. slx9-1 is synthetically lethal with mex67∆loop, mtr2∆loop116-137, or yrb2∆ and strongly synthetically enhanced with rrp12-GFP. Strains containing the indicated WT and mutant alleles were spotted in 10-fold serial dilutions on 5-FOA-SD or SD and grown at 20–30°C for 3–6 days. (B) Slx9 shuttles between the nucleus and the cytoplasm. Cells expressing Enp1-GFP, Gar1-GFP, or Slx9-GFP were mated with kar1-1 cells expressing Nup82-mCherry. The resulting heterokaryons were analyzed by fluorescence microscopy. Scale bar = 5 µm. (C) Slx9 directly binds to RanGTP. GST-Slx9 or GST-Ssb1C was immobilized on GSH-Sepharose before incubating with either 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, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibodies. L = input. (D) Slx9 specifically interacts with the GTP-bound form of Ran. GST-Slx9, GST-Yrb1, or GST-Ntf2 was immobilized on GSH-Sepharose and incubated with buffer alone or 2 µM His6-Ran loaded with GDP or GTP. Analysis of the eluted proteins was carried out as described in (C). L = input. (E) Slx9-1 binding to RanGTP is impaired. Top: GST-Slx9 or GST-Slx9-1 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM His6-RanQLGTP. Analysis of the eluted proteins was carried out as described in (C). L = input. Bottom: bar graph depicts the bound His6-RanQLGTP Western blot signal normalized to GST-Slx9 and GST-Slx9-1 levels, respectively. Four independent experiments were performed and Western blots were quantified by software ImageJ (Version 1.44o). Error bars (S.D.) are indicated.DOI:http://dx.doi.org/10.7554/eLife.05745.004
Mentions: Mutations in MEX67 and MTR2 (mex67Δloop and mtr2Δloop116-137), which encode the essential transport receptor Mex67-Mtr2, are synthetically lethal when combined with the slx9Δ mutant (Faza et al., 2012). In addition, we found that slx9∆ displayed a synthetic growth defect with a strain expressing Rrp12-GFP (Figure 2A). Rrp12 is a 40S pre-ribosome export factor that directly interacts with FG-rich nucleoporins (Oeffinger et al., 2004). Based on these genetic interactions, we asked whether Slx9 functions as a novel export factor for the 40S pre-ribosome. A salient feature of an export factor is that it rapidly shuttles between the nucleus and the cytoplasm. To test this, we employed the established heterokaryon assay (Altvater et al., 2014). WT cells expressing Slx9-GFP were mated to kar1-1 cells, which are deficient in nuclear fusion after cell conjugation, leading to heterokaryon formation. In order to distinguish between the two nuclei, kar1-1 cells also contained Nup82-mCherry as a marker for nuclear pores. As controls, we used the shuttling 40S assembly factor Enp1 and the non-shuttling nucleolar protein Gar1 fused to GFP. Whereas Gar1-GFP was never seen in the nucleus of kar1-1 cells (red signal), both Enp1-GFP and Slx9-GFP localized to both nuclei (Figure 2B). These data are consistent with the shuttling of Slx9 between the nuclear and the cytoplasmic compartments.10.7554/eLife.05745.004Figure 2.Slx9 is a RanGTP binding protein.

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