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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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Related in: MedlinePlus

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.DOI:http://dx.doi.org/10.7554/eLife.05745.003
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fig1: 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.DOI:http://dx.doi.org/10.7554/eLife.05745.003

Mentions: Slx9 is a 24-kDa basic protein that co-enriches with pre-ribosomal particles in the 40S maturation pathway (Gavin et al., 2002; Faza et al., 2012) and is required for efficient nuclear export of 40S pre-ribosomes (Li et al., 2009; Faza et al., 2012). However, the precise contribution of Slx9 to 40S pre-ribosome export has remained unclear. To investigate the function of yeast Slx9, we generated slx9 variants by random mutagenesis and analyzed the growth of the resulting strains at different temperatures. One allele, slx9L108P, hereafter termed slx9-1, caused slow growth at temperatures between 20°C and 30°C, indistinguishable from slx9∆ cells (Figure 1A, top panel). Like slx9∆, slx9-1 cells were not impaired in growth at 37°C (Figure 1A). Western analysis of whole cell lysates revealed that Slx9 and Slx9-1 were present at similar levels (Figure 1A, bottom panel), indicating that impaired growth of the slx9-1 strain is not due to reduced levels of the mutant protein. As previously observed, Slx9-GFP localized primarily to the nucleolus, where it co-localized with the nucleolar marker Gar1-mCherry, as well as to the nucleoplasm (Faza et al., 2012 and Figure 1B). Slx9-1-GFP displayed an identical localization (Figure 1B), indicating that the mutant protein is correctly targeted to the nucleolus and nucleoplasm. Slx9 maximally co-enriched with Enp1-TAP that purifies both the 90S and 40S pre-ribosomes (Faza et al., 2012). A similar purification from slx9-1 cells revealed that Enp1-TAP co-enriched at least as much Slx9-1 mutant protein as Slx9 (Figure 1C). Together, these data show that Slx9-1 is correctly expressed, localized, and recruited to 40S pre-ribosomes.10.7554/eLife.05745.003Figure 1.slx9-1 phenocopies the slx9∆ mutation.


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)

slx9-1 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.DOI:http://dx.doi.org/10.7554/eLife.05745.003
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Related In: Results  -  Collection

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fig1: 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.DOI:http://dx.doi.org/10.7554/eLife.05745.003
Mentions: Slx9 is a 24-kDa basic protein that co-enriches with pre-ribosomal particles in the 40S maturation pathway (Gavin et al., 2002; Faza et al., 2012) and is required for efficient nuclear export of 40S pre-ribosomes (Li et al., 2009; Faza et al., 2012). However, the precise contribution of Slx9 to 40S pre-ribosome export has remained unclear. To investigate the function of yeast Slx9, we generated slx9 variants by random mutagenesis and analyzed the growth of the resulting strains at different temperatures. One allele, slx9L108P, hereafter termed slx9-1, caused slow growth at temperatures between 20°C and 30°C, indistinguishable from slx9∆ cells (Figure 1A, top panel). Like slx9∆, slx9-1 cells were not impaired in growth at 37°C (Figure 1A). Western analysis of whole cell lysates revealed that Slx9 and Slx9-1 were present at similar levels (Figure 1A, bottom panel), indicating that impaired growth of the slx9-1 strain is not due to reduced levels of the mutant protein. As previously observed, Slx9-GFP localized primarily to the nucleolus, where it co-localized with the nucleolar marker Gar1-mCherry, as well as to the nucleoplasm (Faza et al., 2012 and Figure 1B). Slx9-1-GFP displayed an identical localization (Figure 1B), indicating that the mutant protein is correctly targeted to the nucleolus and nucleoplasm. Slx9 maximally co-enriched with Enp1-TAP that purifies both the 90S and 40S pre-ribosomes (Faza et al., 2012). A similar purification from slx9-1 cells revealed that Enp1-TAP co-enriched at least as much Slx9-1 mutant protein as Slx9 (Figure 1C). Together, these data show that Slx9-1 is correctly expressed, localized, and recruited to 40S pre-ribosomes.10.7554/eLife.05745.003Figure 1.slx9-1 phenocopies the slx9∆ mutation.

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