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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 nucleoporin mutants. Analysis of L25-GFP nuclear accumulation in nucleoporin  and ribosome assembly mutants. Haploid double mutants (see  Table I) nup49-313/L25-GFP, nsp1-ala6/L25-GFP nup85::HIS3/ L25-GFP, nup84::HIS3/L25-GFP, and nop1-7/L25-GFP were  grown as described in Materials and Methods in YPD-medium to  OD (600 nm) of 0.2 OD before shift for 14 h to 33°C and reshift  for further 4 h to 20°C. Cells were viewed in the fluorescence microscope to see L25-GFP and under Nomarski optics.
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Figure 7: Nuclear accumulation of L25-GFP in nucleoporin mutants. Analysis of L25-GFP nuclear accumulation in nucleoporin and ribosome assembly mutants. Haploid double mutants (see Table I) nup49-313/L25-GFP, nsp1-ala6/L25-GFP nup85::HIS3/ L25-GFP, nup84::HIS3/L25-GFP, and nop1-7/L25-GFP were grown as described in Materials and Methods in YPD-medium to OD (600 nm) of 0.2 OD before shift for 14 h to 33°C and reshift for further 4 h to 20°C. Cells were viewed in the fluorescence microscope to see L25-GFP and under Nomarski optics.

Mentions: According to these findings, additional nucleoporin mutants were tested in the in vivo ribosomal export assay. Similarly, nsp1-ala6/L25-GFP cells (Fig. 7) and to a lesser extent also nsp1-5/L25-GFP cells (data not shown) revealed an intranuclear accumulation of L25-GFP under the same conditions of incubation as described above. In contrast, neither nup85::HIS3/L25-GFP nor nup84::HIS3/ L25-GFP cells are defective in ribosomal export (Fig. 7), although these double mutant cells are also impaired in growth at 33°C (see also Fig. 2 D). Surprisingly, the nop1-7/ L25-GFP thermosensitive mutant that is inhibited in ribosome biogenesis (Tollervey et al., 1993) did not display a significant nuclear accumulation of L25-GFP; only in a very small number of cells, a spot-like L25-GFP signal was noticed (Fig. 7).


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 nucleoporin mutants. Analysis of L25-GFP nuclear accumulation in nucleoporin  and ribosome assembly mutants. Haploid double mutants (see  Table I) nup49-313/L25-GFP, nsp1-ala6/L25-GFP nup85::HIS3/ L25-GFP, nup84::HIS3/L25-GFP, and nop1-7/L25-GFP were  grown as described in Materials and Methods in YPD-medium to  OD (600 nm) of 0.2 OD before shift for 14 h to 33°C and reshift  for further 4 h to 20°C. Cells were viewed in the fluorescence microscope to see L25-GFP and under Nomarski optics.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132911&req=5

Figure 7: Nuclear accumulation of L25-GFP in nucleoporin mutants. Analysis of L25-GFP nuclear accumulation in nucleoporin and ribosome assembly mutants. Haploid double mutants (see Table I) nup49-313/L25-GFP, nsp1-ala6/L25-GFP nup85::HIS3/ L25-GFP, nup84::HIS3/L25-GFP, and nop1-7/L25-GFP were grown as described in Materials and Methods in YPD-medium to OD (600 nm) of 0.2 OD before shift for 14 h to 33°C and reshift for further 4 h to 20°C. Cells were viewed in the fluorescence microscope to see L25-GFP and under Nomarski optics.
Mentions: According to these findings, additional nucleoporin mutants were tested in the in vivo ribosomal export assay. Similarly, nsp1-ala6/L25-GFP cells (Fig. 7) and to a lesser extent also nsp1-5/L25-GFP cells (data not shown) revealed an intranuclear accumulation of L25-GFP under the same conditions of incubation as described above. In contrast, neither nup85::HIS3/L25-GFP nor nup84::HIS3/ L25-GFP cells are defective in ribosomal export (Fig. 7), although these double mutant cells are also impaired in growth at 33°C (see also Fig. 2 D). Surprisingly, the nop1-7/ L25-GFP thermosensitive mutant that is inhibited in ribosome biogenesis (Tollervey et al., 1993) did not display a significant nuclear accumulation of L25-GFP; only in a very small number of cells, a spot-like L25-GFP signal was noticed (Fig. 7).

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