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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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Ribosomal export in prp20-1 and mutants mapping in  importin/karyopherin β–like transport factors. Thermosensitive  mutants prp20-1, pse1-1/kap123::HIS3, and xpo1-1 were transformed with YEplac195-ADE2-URA3-L25-GFP (pL25-GFP),  or the double xpo1-1/L25-GFP strain was constructed (see Table  I). Cells were grown at 23°C on selective SDC-ura and YPD  plates, respectively, to stationary phase, before they were inoculated in liquid YPD-medium. It was 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 4: Ribosomal export in prp20-1 and mutants mapping in importin/karyopherin β–like transport factors. Thermosensitive mutants prp20-1, pse1-1/kap123::HIS3, and xpo1-1 were transformed with YEplac195-ADE2-URA3-L25-GFP (pL25-GFP), or the double xpo1-1/L25-GFP strain was constructed (see Table I). Cells were grown at 23°C on selective SDC-ura and YPD plates, respectively, to stationary phase, before they were inoculated in liquid YPD-medium. It was 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: Since the L25-GFP signal becomes stronger in cells lacking endogenous L25 (see above), a L25-GFP expressing haploid yeast was constructed in which the rna1-1 ts allele was combined with the chromosomal rpl25::HIS3 gene disruption (see Table I for strain construction, and Fig. 2 B for growth properties). When this rna1-1/L25-GFP strain was shifted for 1 h to 37°C and further incubated for 1 h at 20°C, a strong nuclear L25-GFP accumulation was noticed in many cells. Interestingly, individual cells stained differently showing L25-GFP in the entire nucleus, the nuclear envelope and distinct intranuclear spots, respectively (Fig. 3 B). Again, we noticed that nuclear accumulation of L25-GFP is best seen in the rna1-1 cells grown to prestationary or stationary phase (between OD600nm 4–8) before inoculation in fresh YPD-medium and shift to the restrictive temperature. A lower percentage of cells showed this defect if they were coming from a logarithmically growing culture in YPD-medium (data not shown). It is known that ribosome biogenesis is strongly induced when cells are shifted from stationary/starving conditions into fresh glucose-containing medium (Warner, 1989). This may also enhance nuclear accumulation of L25-GFP. As control served a RNA1+/L25-GFP strain, which did not show under identical conditions any nuclear accumulation of ribosomes after 1 h shift to 37°C and 1 h regrowth at 20°C (Fig. 3 B). The prp20-1 mutant that is defective in nuclear Ran-GDP/ GTP exchange (Kadowaki et al., 1993) was analyzed in a similar way. L25-GFP also accumulates in the nucleus in prp20-1 cells when shifted for 2 h to 37°C followed by regrowth for 40 min at 20°C (Fig. 4). Interestingly, the L25-GFP signal was not evenly distributed in the nucleus, but accumulated in a single or several spots, suggesting a segregation of L25-GFP into distinct subnuclear structures. It was reported earlier that prp20-1 cells are not only altered in mRNA metabolism, but also maintenance of the nuclear structure (Aebi et al., 1990). When the yeast Ran mutants gsp1-1 and gsp1-2 (Wong et al., 1997) expressing L25-GFP were tested, nuclear accumulation of L25-GFP was less prominent as compared with rna1-1 and prp20-1 mutant cells (data not shown). This could mean that not only export, but also nuclear uptake of L25-GFP is significantly impaired in Ran mutants, thus not allowing to detect a clear nuclear accumulation.


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)

Ribosomal export in prp20-1 and mutants mapping in  importin/karyopherin β–like transport factors. Thermosensitive  mutants prp20-1, pse1-1/kap123::HIS3, and xpo1-1 were transformed with YEplac195-ADE2-URA3-L25-GFP (pL25-GFP),  or the double xpo1-1/L25-GFP strain was constructed (see Table  I). Cells were grown at 23°C on selective SDC-ura and YPD  plates, respectively, to stationary phase, before they were inoculated in liquid YPD-medium. It was 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 4: Ribosomal export in prp20-1 and mutants mapping in importin/karyopherin β–like transport factors. Thermosensitive mutants prp20-1, pse1-1/kap123::HIS3, and xpo1-1 were transformed with YEplac195-ADE2-URA3-L25-GFP (pL25-GFP), or the double xpo1-1/L25-GFP strain was constructed (see Table I). Cells were grown at 23°C on selective SDC-ura and YPD plates, respectively, to stationary phase, before they were inoculated in liquid YPD-medium. It was 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: Since the L25-GFP signal becomes stronger in cells lacking endogenous L25 (see above), a L25-GFP expressing haploid yeast was constructed in which the rna1-1 ts allele was combined with the chromosomal rpl25::HIS3 gene disruption (see Table I for strain construction, and Fig. 2 B for growth properties). When this rna1-1/L25-GFP strain was shifted for 1 h to 37°C and further incubated for 1 h at 20°C, a strong nuclear L25-GFP accumulation was noticed in many cells. Interestingly, individual cells stained differently showing L25-GFP in the entire nucleus, the nuclear envelope and distinct intranuclear spots, respectively (Fig. 3 B). Again, we noticed that nuclear accumulation of L25-GFP is best seen in the rna1-1 cells grown to prestationary or stationary phase (between OD600nm 4–8) before inoculation in fresh YPD-medium and shift to the restrictive temperature. A lower percentage of cells showed this defect if they were coming from a logarithmically growing culture in YPD-medium (data not shown). It is known that ribosome biogenesis is strongly induced when cells are shifted from stationary/starving conditions into fresh glucose-containing medium (Warner, 1989). This may also enhance nuclear accumulation of L25-GFP. As control served a RNA1+/L25-GFP strain, which did not show under identical conditions any nuclear accumulation of ribosomes after 1 h shift to 37°C and 1 h regrowth at 20°C (Fig. 3 B). The prp20-1 mutant that is defective in nuclear Ran-GDP/ GTP exchange (Kadowaki et al., 1993) was analyzed in a similar way. L25-GFP also accumulates in the nucleus in prp20-1 cells when shifted for 2 h to 37°C followed by regrowth for 40 min at 20°C (Fig. 4). Interestingly, the L25-GFP signal was not evenly distributed in the nucleus, but accumulated in a single or several spots, suggesting a segregation of L25-GFP into distinct subnuclear structures. It was reported earlier that prp20-1 cells are not only altered in mRNA metabolism, but also maintenance of the nuclear structure (Aebi et al., 1990). When the yeast Ran mutants gsp1-1 and gsp1-2 (Wong et al., 1997) expressing L25-GFP were tested, nuclear accumulation of L25-GFP was less prominent as compared with rna1-1 and prp20-1 mutant cells (data not shown). This could mean that not only export, but also nuclear uptake of L25-GFP is significantly impaired in Ran mutants, thus not allowing to detect a clear nuclear accumulation.

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