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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 nup49-313 ts mutant. (A) Time-course of nuclear accumulation of L25-GFP in  nup49-313/L25-GFP cells. Cells were first grown at 23°C before  shift for 14 h to 33°C. After the restrictive growth condition, cells  were incubated at the permissive temperature (20°C) and inspected in the fluorescence microscope after 0, 1, 4, 10, and 24 h.  (B) Indirect immunofluorescence microscopy of nup49-313/L25-GFP cells. After fixation of cells in 3.7% formaldehyde, spheroplasted cells were processed for indirect immunofluorescence using anti-Pus1p antibodies. Cells were also stained for DNA with  Hoechst 33258. The L25-GFP signal was recorded in the fluorescein channel, the Pus1p immunostaining in the rhodamin channel  of a Zeiss Axioskop fluorescence microscope.
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Figure 6: Nuclear accumulation of L25-GFP in nup49-313 ts mutant. (A) Time-course of nuclear accumulation of L25-GFP in nup49-313/L25-GFP cells. Cells were first grown at 23°C before shift for 14 h to 33°C. After the restrictive growth condition, cells were incubated at the permissive temperature (20°C) and inspected in the fluorescence microscope after 0, 1, 4, 10, and 24 h. (B) Indirect immunofluorescence microscopy of nup49-313/L25-GFP cells. After fixation of cells in 3.7% formaldehyde, spheroplasted cells were processed for indirect immunofluorescence using anti-Pus1p antibodies. Cells were also stained for DNA with Hoechst 33258. The L25-GFP signal was recorded in the fluorescein channel, the Pus1p immunostaining in the rhodamin channel of a Zeiss Axioskop fluorescence microscope.

Mentions: Since the onset of the ts phenotype in the various nucleoporin and nop1 mutants requires several hours of incubation at the nonpermissive temperature (Nehrbass et al., 1993; Doye et al., 1994; Siniossoglou et al., 1996), ts strains were shifted for longer than 6 h to the restrictive growth condition (see also Doye et al., 1994; Nehrbass et al., 1993), before regrowth at the permissive temperature to allow for de novo ribosome biogenesis (see also Fig. 3). A time course was performed to follow the onset of a ribosomal export defect in the nup49-313/L25-GFP strain (Fig. 6 A). The minimal time required to observe L25-GFP nuclear mislocation in nup49-313 cells was to shift for 7 h to 33°C, but strongest accumulation was seen between 10– 14 h shift to 33°C. For reasons of convenience, we incubated over night at 33°C (i.e., 14 h), before regrowth at 20°C was induced. Already after 1 h incubation of nup49-313/L25-GFP cells at permissive conditions, nuclear L25-GFP accumulation became apparent, which increased within the next 3 h of incubation (Fig. 6 A). Upon longer growth at 20°C (>10 h), cells progressively lost their nuclear L25-GFP signal and cytoplasmic labeling became stronger. During the first 10 h of growth at 20°C, the optical density of the nup49-313/L25-GFP cell culture increased continuously showing that the cells indeed regrew (data not shown). Such an incubation scheme did not impair ribosomal export at all in the L25-GFP control strain that exhibits an exclusive cytoplasmic location of L25-GFP with no nuclear accumulation under all tested conditions (data not shown). To verify that L25-GFP accumulated inside the nucleus in nup49-313 cells, indirect immunofluorescence microscopy using anti-Pus1p antibodies (a nuclear marker protein; Simos et al., 1996b), and DNA staining was performed. Clearly, the L25-GFP signal colocalized with the Pus1p immunolabeling and partly with the DNA staining (Fig. 6 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)

Nuclear accumulation of L25-GFP in nup49-313 ts mutant. (A) Time-course of nuclear accumulation of L25-GFP in  nup49-313/L25-GFP cells. Cells were first grown at 23°C before  shift for 14 h to 33°C. After the restrictive growth condition, cells  were incubated at the permissive temperature (20°C) and inspected in the fluorescence microscope after 0, 1, 4, 10, and 24 h.  (B) Indirect immunofluorescence microscopy of nup49-313/L25-GFP cells. After fixation of cells in 3.7% formaldehyde, spheroplasted cells were processed for indirect immunofluorescence using anti-Pus1p antibodies. Cells were also stained for DNA with  Hoechst 33258. The L25-GFP signal was recorded in the fluorescein channel, the Pus1p immunostaining in the rhodamin channel  of a Zeiss Axioskop fluorescence microscope.
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Figure 6: Nuclear accumulation of L25-GFP in nup49-313 ts mutant. (A) Time-course of nuclear accumulation of L25-GFP in nup49-313/L25-GFP cells. Cells were first grown at 23°C before shift for 14 h to 33°C. After the restrictive growth condition, cells were incubated at the permissive temperature (20°C) and inspected in the fluorescence microscope after 0, 1, 4, 10, and 24 h. (B) Indirect immunofluorescence microscopy of nup49-313/L25-GFP cells. After fixation of cells in 3.7% formaldehyde, spheroplasted cells were processed for indirect immunofluorescence using anti-Pus1p antibodies. Cells were also stained for DNA with Hoechst 33258. The L25-GFP signal was recorded in the fluorescein channel, the Pus1p immunostaining in the rhodamin channel of a Zeiss Axioskop fluorescence microscope.
Mentions: Since the onset of the ts phenotype in the various nucleoporin and nop1 mutants requires several hours of incubation at the nonpermissive temperature (Nehrbass et al., 1993; Doye et al., 1994; Siniossoglou et al., 1996), ts strains were shifted for longer than 6 h to the restrictive growth condition (see also Doye et al., 1994; Nehrbass et al., 1993), before regrowth at the permissive temperature to allow for de novo ribosome biogenesis (see also Fig. 3). A time course was performed to follow the onset of a ribosomal export defect in the nup49-313/L25-GFP strain (Fig. 6 A). The minimal time required to observe L25-GFP nuclear mislocation in nup49-313 cells was to shift for 7 h to 33°C, but strongest accumulation was seen between 10– 14 h shift to 33°C. For reasons of convenience, we incubated over night at 33°C (i.e., 14 h), before regrowth at 20°C was induced. Already after 1 h incubation of nup49-313/L25-GFP cells at permissive conditions, nuclear L25-GFP accumulation became apparent, which increased within the next 3 h of incubation (Fig. 6 A). Upon longer growth at 20°C (>10 h), cells progressively lost their nuclear L25-GFP signal and cytoplasmic labeling became stronger. During the first 10 h of growth at 20°C, the optical density of the nup49-313/L25-GFP cell culture increased continuously showing that the cells indeed regrew (data not shown). Such an incubation scheme did not impair ribosomal export at all in the L25-GFP control strain that exhibits an exclusive cytoplasmic location of L25-GFP with no nuclear accumulation under all tested conditions (data not shown). To verify that L25-GFP accumulated inside the nucleus in nup49-313 cells, indirect immunofluorescence microscopy using anti-Pus1p antibodies (a nuclear marker protein; Simos et al., 1996b), and DNA staining was performed. Clearly, the L25-GFP signal colocalized with the Pus1p immunolabeling and partly with the DNA staining (Fig. 6 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