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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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Nuclear accumulation of L25-GFP in rna1-1  mutant. Thermosensitive  mutant rna1-1 transformed  with YEplac195-ADE2-URA3-L25-GFP (A), and  the rpl25::HIS3 disruption  mutant rna1-1/L25-GFP (B)  were used. Cells were grown  at 23°C on selective SDC-ura  plates (A) and YPD plates  (B) to stationary phase (3–4 d  on plate) before they were  inoculated in liquid YPD-medium. It was then shifted  for the indicated time points  to 37°C, before cells were  further incubated at room  temperature (20°C). After  centrifugation, cells were resuspended in water, mounted  on a slide and inspected in  the fluorescence microscope.
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Figure 3: Nuclear accumulation of L25-GFP in rna1-1 mutant. Thermosensitive mutant rna1-1 transformed with YEplac195-ADE2-URA3-L25-GFP (A), and the rpl25::HIS3 disruption mutant rna1-1/L25-GFP (B) were used. Cells were grown at 23°C on selective SDC-ura plates (A) and YPD plates (B) to stationary phase (3–4 d on plate) before they were inoculated in liquid YPD-medium. It was then shifted for the indicated time points to 37°C, before cells were further incubated at room temperature (20°C). After centrifugation, cells were resuspended in water, mounted on a slide and inspected in the fluorescence microscope.

Mentions: The small GTPase Ran has been suggested to be a universal regulator of nucleocytoplasmic transport (Koepp and Silver, 1996). By exploiting the L25-GFP assay, we tested whether ribosomal export is sensitive to perturbations of the Ran-GTP/GDP cycle. Therefore, the Ran-GAP mutant rna1-1 (Hopper et al., 1978; Bischoff et al., 1995) and the Ran-GEF mutant prp20-1 (Aebi et al., 1990; Amberg et al., 1993; Kadowaki et al., 1993) were transformed with the L25-GFP reporter construct. However, no nuclear accumulation of L25-GFP was seen when the rna1-1 cells were grown at the permissive temperature (23°C) and shifted for various time points to 37°C (Fig. 3 A). Since it is known that rRNA transcription and ribosome biogenesis is downregulated in a very complex way in yeast upon nutritional, mutational, and temperature stress (Warner, 1989; Woolford, 1991), the in vivo assay was performed differently. Ts mutants derived from prestationary or stationary culture conditions were first shifted to the restrictive temperature to induce the mutant phenotype and then incubated at the permissive condition to reinduce de novo ribosome biogenesis. Under these conditions, nuclear accumulation of the L25-GFP reporter was seen in many rna1-1 mutant cells (Fig. 3 A). The nuclear L25-GFP signal became visible already after 1 h shift to 37°C and 30 min regrowth at 20°C (data not shown), and developed stronger after 2 h at 37°C and 30 min induction at 20°C (Fig. 3 A). After prolonged permissive growth conditions (180 min), nuclear accumulation of L25-GFP vanished in most of the cells; interestingly, some cells showed a distinct nuclear envelope staining at this later time point (Fig. 3 A). A RNA1+ control strain exhibits under all tested conditions an exclusive cytoplasmic L25-GFP location with nuclear exclusion (see also later).


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)

Nuclear accumulation of L25-GFP in rna1-1  mutant. Thermosensitive  mutant rna1-1 transformed  with YEplac195-ADE2-URA3-L25-GFP (A), and  the rpl25::HIS3 disruption  mutant rna1-1/L25-GFP (B)  were used. Cells were grown  at 23°C on selective SDC-ura  plates (A) and YPD plates  (B) to stationary phase (3–4 d  on plate) before they were  inoculated in liquid YPD-medium. It was then shifted  for the indicated time points  to 37°C, before cells were  further incubated at room  temperature (20°C). After  centrifugation, cells were resuspended in water, mounted  on a slide and inspected in  the fluorescence microscope.
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Related In: Results  -  Collection

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Figure 3: Nuclear accumulation of L25-GFP in rna1-1 mutant. Thermosensitive mutant rna1-1 transformed with YEplac195-ADE2-URA3-L25-GFP (A), and the rpl25::HIS3 disruption mutant rna1-1/L25-GFP (B) were used. Cells were grown at 23°C on selective SDC-ura plates (A) and YPD plates (B) to stationary phase (3–4 d on plate) before they were inoculated in liquid YPD-medium. It was then shifted for the indicated time points to 37°C, before cells were further incubated at room temperature (20°C). After centrifugation, cells were resuspended in water, mounted on a slide and inspected in the fluorescence microscope.
Mentions: The small GTPase Ran has been suggested to be a universal regulator of nucleocytoplasmic transport (Koepp and Silver, 1996). By exploiting the L25-GFP assay, we tested whether ribosomal export is sensitive to perturbations of the Ran-GTP/GDP cycle. Therefore, the Ran-GAP mutant rna1-1 (Hopper et al., 1978; Bischoff et al., 1995) and the Ran-GEF mutant prp20-1 (Aebi et al., 1990; Amberg et al., 1993; Kadowaki et al., 1993) were transformed with the L25-GFP reporter construct. However, no nuclear accumulation of L25-GFP was seen when the rna1-1 cells were grown at the permissive temperature (23°C) and shifted for various time points to 37°C (Fig. 3 A). Since it is known that rRNA transcription and ribosome biogenesis is downregulated in a very complex way in yeast upon nutritional, mutational, and temperature stress (Warner, 1989; Woolford, 1991), the in vivo assay was performed differently. Ts mutants derived from prestationary or stationary culture conditions were first shifted to the restrictive temperature to induce the mutant phenotype and then incubated at the permissive condition to reinduce de novo ribosome biogenesis. Under these conditions, nuclear accumulation of the L25-GFP reporter was seen in many rna1-1 mutant cells (Fig. 3 A). The nuclear L25-GFP signal became visible already after 1 h shift to 37°C and 30 min regrowth at 20°C (data not shown), and developed stronger after 2 h at 37°C and 30 min induction at 20°C (Fig. 3 A). After prolonged permissive growth conditions (180 min), nuclear accumulation of L25-GFP vanished in most of the cells; interestingly, some cells showed a distinct nuclear envelope staining at this later time point (Fig. 3 A). A RNA1+ control strain exhibits under all tested conditions an exclusive cytoplasmic L25-GFP location with nuclear exclusion (see also later).

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