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A novel in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin mutants.

Hurt E, Hannus S, Schmelzl B, Lau D, Tollervey D, Simos G - J. Cell Biol. (1999)

Bottom Line: However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP.Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export.Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins.

View Article: PubMed Central - PubMed

Affiliation: Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

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NLS-mediated nuclear protein uptake and  poly(A)+ RNA export in  thermosensitive nup49-313  cells. (A) The thermosensitive nup49-313 mutant was  transformed with pGAL-NLS-GFP-lacZ (classical  NLS of SV-40 large T antigen) and transformants were  selected on SDC (−ura)  plates. Transformants were  grown in selective synthetic  raffinose (−ura) medium at  20°C. One-half of the culture  was left at 20°C (right panel),  the other half was shifted for  11 h to 37°C (left panel).  Cells from both cultures  were collected by centrifugation, transfered into in SGal  (−ura) medium and incubated for 5 h at 20°C to induce pGAL-NLS-GFP-lacZ expression. Cells were finally inspected in the fluorescence microscope. Note that  at both temperatures the nuclear import reporter accumulates in the nucleus. (B) Nuclear export of poly(A)+ RNA in nup49-313/L25-GFP and L25-GFP cells. Cells were grown at the indicated temperatures (14 h at 33°C; 14 h at 33°C, and 4 h at 20°C; 18 h at 20°C) before  they were fixed and processed for in situ hybridization using a fluorescently labeled oligo dT-probe.
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Figure 8: NLS-mediated nuclear protein uptake and poly(A)+ RNA export in thermosensitive nup49-313 cells. (A) The thermosensitive nup49-313 mutant was transformed with pGAL-NLS-GFP-lacZ (classical NLS of SV-40 large T antigen) and transformants were selected on SDC (−ura) plates. Transformants were grown in selective synthetic raffinose (−ura) medium at 20°C. One-half of the culture was left at 20°C (right panel), the other half was shifted for 11 h to 37°C (left panel). Cells from both cultures were collected by centrifugation, transfered into in SGal (−ura) medium and incubated for 5 h at 20°C to induce pGAL-NLS-GFP-lacZ expression. Cells were finally inspected in the fluorescence microscope. Note that at both temperatures the nuclear import reporter accumulates in the nucleus. (B) Nuclear export of poly(A)+ RNA in nup49-313/L25-GFP and L25-GFP cells. Cells were grown at the indicated temperatures (14 h at 33°C; 14 h at 33°C, and 4 h at 20°C; 18 h at 20°C) before they were fixed and processed for in situ hybridization using a fluorescently labeled oligo dT-probe.

Mentions: Apparently, nuclear uptake of L25-GFP still proceeds in nup49-313 cells (as well as in nsp1-ala6, rna1-1, and prp20-1 cells) after shift from restrictive to permissive growth conditions. Therefore, we tested other nucleocytoplasmic transport reactions. A galactose-inducible GAL-NLS-GFP-lacZ construct was transformed into the nup49-313 mutant and the culture was shifted for 11 h to 37°C under conditions of promoter repression (raffinose medium). Then, cells were transferred into galactose-containing medium to induce pGAL-NLS-GFP-lacZ and incubated for 5 h at 20°C. When cells were inspected in the fluorescence microscope, NLS-GFP-lacZ still accumulated inside the nucleus, although also a weak cytoplasmic staining could be seen in a few cells (Fig. 8 A). This shows that nuclear accumulation of this NLS-reporter can occur under conditions in which ribosomal export is apparently impaired (see also Figs. 6 and 7). When the distribution of poly(A)+ RNA was examined in the nup49-313/L25-GFP mutant, nuclear mRNA accumulation was seen in ∼10–20% of the cells when incubated at 33°C, and this percentage further increased when cells were brought back from the restrictive to permissive condition (Fig. 8 B). No mRNA export defect was observed in the L25-GFP strain grown at the various temperatures (Fig. 8 B).


A novel in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin mutants.

Hurt E, Hannus S, Schmelzl B, Lau D, Tollervey D, Simos G - J. Cell Biol. (1999)

NLS-mediated nuclear protein uptake and  poly(A)+ RNA export in  thermosensitive nup49-313  cells. (A) The thermosensitive nup49-313 mutant was  transformed with pGAL-NLS-GFP-lacZ (classical  NLS of SV-40 large T antigen) and transformants were  selected on SDC (−ura)  plates. Transformants were  grown in selective synthetic  raffinose (−ura) medium at  20°C. One-half of the culture  was left at 20°C (right panel),  the other half was shifted for  11 h to 37°C (left panel).  Cells from both cultures  were collected by centrifugation, transfered into in SGal  (−ura) medium and incubated for 5 h at 20°C to induce pGAL-NLS-GFP-lacZ expression. Cells were finally inspected in the fluorescence microscope. Note that  at both temperatures the nuclear import reporter accumulates in the nucleus. (B) Nuclear export of poly(A)+ RNA in nup49-313/L25-GFP and L25-GFP cells. Cells were grown at the indicated temperatures (14 h at 33°C; 14 h at 33°C, and 4 h at 20°C; 18 h at 20°C) before  they were fixed and processed for in situ hybridization using a fluorescently labeled oligo dT-probe.
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Related In: Results  -  Collection

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Figure 8: NLS-mediated nuclear protein uptake and poly(A)+ RNA export in thermosensitive nup49-313 cells. (A) The thermosensitive nup49-313 mutant was transformed with pGAL-NLS-GFP-lacZ (classical NLS of SV-40 large T antigen) and transformants were selected on SDC (−ura) plates. Transformants were grown in selective synthetic raffinose (−ura) medium at 20°C. One-half of the culture was left at 20°C (right panel), the other half was shifted for 11 h to 37°C (left panel). Cells from both cultures were collected by centrifugation, transfered into in SGal (−ura) medium and incubated for 5 h at 20°C to induce pGAL-NLS-GFP-lacZ expression. Cells were finally inspected in the fluorescence microscope. Note that at both temperatures the nuclear import reporter accumulates in the nucleus. (B) Nuclear export of poly(A)+ RNA in nup49-313/L25-GFP and L25-GFP cells. Cells were grown at the indicated temperatures (14 h at 33°C; 14 h at 33°C, and 4 h at 20°C; 18 h at 20°C) before they were fixed and processed for in situ hybridization using a fluorescently labeled oligo dT-probe.
Mentions: Apparently, nuclear uptake of L25-GFP still proceeds in nup49-313 cells (as well as in nsp1-ala6, rna1-1, and prp20-1 cells) after shift from restrictive to permissive growth conditions. Therefore, we tested other nucleocytoplasmic transport reactions. A galactose-inducible GAL-NLS-GFP-lacZ construct was transformed into the nup49-313 mutant and the culture was shifted for 11 h to 37°C under conditions of promoter repression (raffinose medium). Then, cells were transferred into galactose-containing medium to induce pGAL-NLS-GFP-lacZ and incubated for 5 h at 20°C. When cells were inspected in the fluorescence microscope, NLS-GFP-lacZ still accumulated inside the nucleus, although also a weak cytoplasmic staining could be seen in a few cells (Fig. 8 A). This shows that nuclear accumulation of this NLS-reporter can occur under conditions in which ribosomal export is apparently impaired (see also Figs. 6 and 7). When the distribution of poly(A)+ RNA was examined in the nup49-313/L25-GFP mutant, nuclear mRNA accumulation was seen in ∼10–20% of the cells when incubated at 33°C, and this percentage further increased when cells were brought back from the restrictive to permissive condition (Fig. 8 B). No mRNA export defect was observed in the L25-GFP strain grown at the various temperatures (Fig. 8 B).

Bottom Line: However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP.Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export.Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins.

View Article: PubMed Central - PubMed

Affiliation: Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

Show MeSH
Related in: MedlinePlus