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 …
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 …
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-RanQLGTPRK…
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: …
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). …
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). …
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 …
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: …
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 …
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 …
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 …
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
