A non-canonical mechanism for Crm1-export cargo complex assembly
- ETH Zurich, Switzerland
- Graduate School, Switzerland
Figures
Figure 1
slx9-1 phenocopies the slx9∆ mutation.
(A) The slx9-1 allele does not complement the slow growth of slx9∆ cells. Top: SLX9, slx9∆, and slx9-1 cells were spotted in 10-fold dilutions on SD-plates and grown at the indicated temperatures for 3–6 days. Bottom: Slx9 protein levels from whole cell extracts derived from the indicated strains were determined by Western analysis using antibodies directed against Slx9. Levels of the protein Arc1 served as a loading control. (B) Slx9-1 localizes to the nucleolus/nucleoplasm. Cells expressing Gar1-mCherry and Slx9-GFP or Slx9-1-GFP were grown until mid-log phase. Localization of the indicated fusion proteins was analyzed by fluorescence microscopy. Gar1-mCherry served as a nucleolar marker. Scale bar = 5 µm. (C) Slx9-1 is recruited to the early 40S pre-ribosome. Enp1-TAP was isolated by tandem affinity purification (TAP) from the indicated strains. Calmodulin-eluates were separated on a 4–12% gradient gel and analyzed by either silver staining or Western using the indicated antibodies. The ribosomal protein uS7 served as a loading control. (D) slx9-1 cells are impaired in nuclear export of 40S pre-ribosomes. Top: localization of uS5-GFP was monitored by fluorescence microscopy. Bottom: localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained by DAPI (blue). Scale bar = 5 µm.
Figure 2
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.
Figure 3
The basic patch and acidic tail of Ran modulates interactions with Slx9.
(A) The basic patch of RanQLGTP contributes to Slx9 binding. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP, His6-RanQLGTPRKAA, or His6-RanQLGTPRKEE). 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 antibody. L = input. (B) The acidic tail of RanQLGTP negatively regulates interactions with Slx9. GST-Slx9, GST-Yrb1, or GST-Kap123 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM Ran (His6-RanQLGTP or His6-Ran∆CQLGTP). Analysis of the eluted proteins was carried out as described in (A). L = input.
Figure 4
Slx9 directly binds the 40S pre-ribosome nuclear export signal (NES)-containing adaptor Rio2 and RanQLGTP.
(A) Rio2 and Ltv1 export complex formation requires their C-terminal NESs. Top: the positions of the Rio2 and Ltv1 NESs are shown. Hydrophobic residues in these NESs are highlighted in red. Bottom: GST-Rio2, GST-Rio2∆NES, GST-Ltv1, or GST-Ltv1∆NES was immobilized on GSH-Sepharose, and complex formation was analyzed as in Figure 2C. L = input. (B) Slx9 directly interacts with Rio2. Immobilized GST-Rio2 or GST-Ltv1 was incubated with buffer alone or 0.5 µM Slx9. Conversely, immobilized GST-Slx9 was incubated with buffer alone or with lysate containing His6-Nmd3 or His6-Rio2. Analysis of the eluted proteins was carried out as described in Figure 2C. L = input.
Figure 5
Slx9 binds to Rio2 and RanGTP using distinct binding surfaces.
(A) GST-Rio2 was immobilized on GSH-Sepharose and incubated with buffer, 2 µM His6-RanQLGTP, or 0.5 µM Slx9 (+1). After washing, the GST-Rio2:Slx9 complex was incubated with 2 µM His6-RanQLGTP (+2). Analysis of the eluted proteins was carried out as described in Figure 2C. L = input. (B) RanGTP does not displace Slx9 from a preformed GST-Rio2:Slx9 complex. Top: immobilized GST-Rio2 was incubated with buffer or increasing concentrations of His6-RanQLGTP (62.5 nM–32 µM). Bottom: immobilized GST-Rio2 was incubated with either buffer or 1 µM Slx9. The unbound Slx9 was washed away, and the resulting GST-Rio2:Slx9 complex was incubated with increasing concentrations of His6-RanQLGTP (62.5 nM–32 µM). Analysis of the eluted proteins was carried out as described in Figure 2C. L = input. (C) RanQLGTP does not displace Rio2 from a preformed GST-Slx9:Rio2 complex. Top: immobilized GST-Slx9 was incubated with buffer or increasing concentrations of His6-RanQLGTP (62.5 nM–32 µM). Bottom: immobilized GST-Slx9 was incubated with excess of Rio2. The unbound Rio2 was washed away, and the resulting complex GST-Slx9:Rio2 complex was incubated with increasing concentrations of His6-RanQLGTP (62.5 nM–32 µM). Analysis of the eluted proteins was carried out as described in Figure 2C. L = input.
Figure 6
Slx9 promotes stepwise assembly of a Crm1-export complex on the NES of Rio2.
(A) Flow chart depicting the experimental setup to assemble a Rio2:Slx9:RanQLGTP:Crm1 complex. Immobilized GST-Rio2 was sequentially incubated with Slx9 (red), RanQLGTP (purple), and Crm1 (green). Unbound protein was washed away after each incubation step. (B) Crm1 is recruited to the GST-Rio2:Slx9:RanGTP complex in a NES-dependent manner. Immobilized GST-Rio2 or GST-Rio2∆NES was incubated with buffer alone or 0.5 µM Slx9, followed by the stepwise addition of 0.2 µM His6-RanQLGTP and 50 nM Crm1-His6, as depicted in (A). After a final washing step, bound proteins were analyzed as in Figure 2C. L = input. (C) Crm1 is not recruited to the GST-Rio2:Slx9 complex. Immobilized GST-Rio2 was incubated with buffer alone or 0.5 µM Slx9, followed by addition of buffer, 50 nM Crm1-His6, or the stepwise addition of 0.2 µM His6-RanQLGTP and 50 nM Crm1-His6 as depicted in (A). Analysis of the bound proteins was carried out as described in Figure 2C. L = input. (D) Recruitment of Crm1 to a Rio2:Slx9-1:RanQLGTP complex is impaired. Immobilized GST-Rio2 was incubated with buffer alone, 0.5 µM Slx9 or 0.5 µM Slx9-1, followed by the stepwise addition of 0.2 µM His6-RanQLGTP and 50 nM Crm1-His6 as depicted in (A). Analysis of the bound proteins was carried out as described in Figure 2C. L = input.
Figure 7 with 2 supplements
Slx9 provides a scaffold to load Crm1 onto Rio2-NES.
(A) Rio23G does not interact with Crm1 in the presence of RanGTP. Top: schematic depicts the positions of mutations proximal to the NES (399-EEN-401-GGG) in the Rio23G. Hydrophobic amino acids of the NES are red and mutated amino acids are orange. Bottom: GST-Rio23G was immobilized on GSH-Sepharose and binding reactions were carried out and analyzed as in Figure 2C. L = input. (B) rio2∆NES, but not rio23G, is synthetically lethal with mex67∆loop and 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. (C) Slx9 restores Crm1 binding to the Rio23G:Slx9:RanQLGTP complex. GST-Rio2:Slx9:RanQLGTP or GST-Rio23G:Slx9:RanQLGTP was incubated with buffer alone or 50 nM Crm1-His6. Bound proteins were analyzed as in Figure 2C. L = input. (D) Crm1 is impaired in binding a Rio23G:Slx9-1:RanQLGTP complex. Immobilized GST-Rio23G was incubated with buffer alone, 0.5 µM Slx9 or 0.5 µM Slx9-1, followed by the stepwise addition of 0.2 µM His6-RanQLGTP and 50 nM Crm1-His6 as depicted in (A). Analysis of the bound proteins was carried out as described in Figure 2C. L = input.
Figure 7—figure supplement 1
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.
Figure 7—figure supplement 2
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.
Figure 8
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.
Author response image 1
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.
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.05745.013
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Supplementary file 2
Plasmids used in this study.
- https://doi.org/10.7554/eLife.05745.014
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A non-canonical mechanism for Crm1-export cargo complex assembly
eLife 4:e05745.
https://doi.org/10.7554/eLife.05745
