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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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L25-GFP containing ribosomes isolated from nup49-313/L25-GFP and Gsp1p-G21V/L25-GFP cells. Ribosomes were  isolated under low salt conditions (100 mM KCl) from (A)  nup49-313/L25-GFP cells shifted for 14 h to 33°C and incubated  for another 4 h at 20°C and (B) L25-GFP cells expressing Gsp1p-G21V under restrictive conditions (i.e., galactose medium) as described in the legend of Fig. 5 and in Materials and Methods.  Whole cell extracts were prepared, loaded on a 10–40% sucrose  gradient and centrifuged for 12 h at 150,000 g. The fractions from  the sucrose density gradient were TCA-precipitated, resuspended in SDS-sample buffer and analyzed by SDS-PAGE and  Coomassie-staining (upper part) or Western blotting using anti-GFP antibodies (lower part). The position of L25-GFP is indicated. The asterisks mark the position of prominent ribosomal  proteins. Marker proteins (10-kD ladder with a stronger 50-kD  band) are also depicted.
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Figure 9: L25-GFP containing ribosomes isolated from nup49-313/L25-GFP and Gsp1p-G21V/L25-GFP cells. Ribosomes were isolated under low salt conditions (100 mM KCl) from (A) nup49-313/L25-GFP cells shifted for 14 h to 33°C and incubated for another 4 h at 20°C and (B) L25-GFP cells expressing Gsp1p-G21V under restrictive conditions (i.e., galactose medium) as described in the legend of Fig. 5 and in Materials and Methods. Whole cell extracts were prepared, loaded on a 10–40% sucrose gradient and centrifuged for 12 h at 150,000 g. The fractions from the sucrose density gradient were TCA-precipitated, resuspended in SDS-sample buffer and analyzed by SDS-PAGE and Coomassie-staining (upper part) or Western blotting using anti-GFP antibodies (lower part). The position of L25-GFP is indicated. The asterisks mark the position of prominent ribosomal proteins. Marker proteins (10-kD ladder with a stronger 50-kD band) are also depicted.

Mentions: To analyze whether L25-GFP is associated with ribosomes under conditions in which it accumulates inside the nucleus (see also Figs. 6 and 7), a whole cell extract was prepared from nup49-313/L25-GFP cells and ribosomes and soluble proteins were separated by sucrose gradient centrifugation. When the various sucrose gradient fractions were tested by Western blotting using anti-GFP antibodies, no free L25-GFP was detected in the upper part of the gradient, but L25-GFP was exclusively recovered in the lower gradient fractions in which the ribosomes migrated (Fig. 9 A, compare the Coomassie pattern of the major ribosomal proteins with the L25-GFP signal). This suggests that the L25-GFP that has accumulated in the nucleus is part of assembled ribosomes. As described above, free L25-GFP could be detected in the upper fractions of the sucrose density gradient, when authentic L25 was coexpressed (see Fig. 1 C). Ribosomes were also isolated from the strain L25-GFP that expresses dominant-negative Gsp1p-G21V (see also Fig. 4) under galactose-inducing conditions. All L25-GFP cofractioned with the ribosomal peak fractions and no free L25-GFP was found in the upper part of the sucrose gradient (Fig. 9 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)

L25-GFP containing ribosomes isolated from nup49-313/L25-GFP and Gsp1p-G21V/L25-GFP cells. Ribosomes were  isolated under low salt conditions (100 mM KCl) from (A)  nup49-313/L25-GFP cells shifted for 14 h to 33°C and incubated  for another 4 h at 20°C and (B) L25-GFP cells expressing Gsp1p-G21V under restrictive conditions (i.e., galactose medium) as described in the legend of Fig. 5 and in Materials and Methods.  Whole cell extracts were prepared, loaded on a 10–40% sucrose  gradient and centrifuged for 12 h at 150,000 g. The fractions from  the sucrose density gradient were TCA-precipitated, resuspended in SDS-sample buffer and analyzed by SDS-PAGE and  Coomassie-staining (upper part) or Western blotting using anti-GFP antibodies (lower part). The position of L25-GFP is indicated. The asterisks mark the position of prominent ribosomal  proteins. Marker proteins (10-kD ladder with a stronger 50-kD  band) are also depicted.
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Related In: Results  -  Collection

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Figure 9: L25-GFP containing ribosomes isolated from nup49-313/L25-GFP and Gsp1p-G21V/L25-GFP cells. Ribosomes were isolated under low salt conditions (100 mM KCl) from (A) nup49-313/L25-GFP cells shifted for 14 h to 33°C and incubated for another 4 h at 20°C and (B) L25-GFP cells expressing Gsp1p-G21V under restrictive conditions (i.e., galactose medium) as described in the legend of Fig. 5 and in Materials and Methods. Whole cell extracts were prepared, loaded on a 10–40% sucrose gradient and centrifuged for 12 h at 150,000 g. The fractions from the sucrose density gradient were TCA-precipitated, resuspended in SDS-sample buffer and analyzed by SDS-PAGE and Coomassie-staining (upper part) or Western blotting using anti-GFP antibodies (lower part). The position of L25-GFP is indicated. The asterisks mark the position of prominent ribosomal proteins. Marker proteins (10-kD ladder with a stronger 50-kD band) are also depicted.
Mentions: To analyze whether L25-GFP is associated with ribosomes under conditions in which it accumulates inside the nucleus (see also Figs. 6 and 7), a whole cell extract was prepared from nup49-313/L25-GFP cells and ribosomes and soluble proteins were separated by sucrose gradient centrifugation. When the various sucrose gradient fractions were tested by Western blotting using anti-GFP antibodies, no free L25-GFP was detected in the upper part of the gradient, but L25-GFP was exclusively recovered in the lower gradient fractions in which the ribosomes migrated (Fig. 9 A, compare the Coomassie pattern of the major ribosomal proteins with the L25-GFP signal). This suggests that the L25-GFP that has accumulated in the nucleus is part of assembled ribosomes. As described above, free L25-GFP could be detected in the upper fractions of the sucrose density gradient, when authentic L25 was coexpressed (see Fig. 1 C). Ribosomes were also isolated from the strain L25-GFP that expresses dominant-negative Gsp1p-G21V (see also Fig. 4) under galactose-inducing conditions. All L25-GFP cofractioned with the ribosomal peak fractions and no free L25-GFP was found in the upper part of the sucrose gradient (Fig. 9 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