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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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Accumulation of  L25-GFP inside the nucleus  in strains overexpressing  Los1p and Gsp1p-G21V.  L25-GFP cells carrying the  plasmids (A) pEMBLyex4  (GAL), (B) pGAL-GSP1-G21V, (C) pGAL-KAP95,  (D) pGAL-YRB4ΔN,  (E) pEMBLyex4-ProtA-LOS1 (GAL-LOS1), (F  and G) pEMBLyex4-ProtA-LOS1ΔN (GAL-LOS1ΔN)  were first grown in raffinose-containing medium before  transferring them into galactose-containing medium/ plate (see in Materials and  Methods). After growth in  galactose for 12 h, the localization of L25-GFP was determined in the fluorescence  microscope (A–F). GAL-LOS1ΔN cells were also  fixed in 3.7% formaldehyde  for 30 min before spheroplasting and indirect immunofluorescence microscopy (G).  The L25-GFP signal (fluorescein channel; indicated by arrows), the Nop1p signal  (rhodamin channel) and the  DNA signal (Hoechst channel) were recorded in a Zeiss  Axioskop fluorescence microscope. L25-GFP and  Nop1p colocation is indicated by arrows.
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Figure 5: Accumulation of L25-GFP inside the nucleus in strains overexpressing Los1p and Gsp1p-G21V. L25-GFP cells carrying the plasmids (A) pEMBLyex4 (GAL), (B) pGAL-GSP1-G21V, (C) pGAL-KAP95, (D) pGAL-YRB4ΔN, (E) pEMBLyex4-ProtA-LOS1 (GAL-LOS1), (F and G) pEMBLyex4-ProtA-LOS1ΔN (GAL-LOS1ΔN) were first grown in raffinose-containing medium before transferring them into galactose-containing medium/ plate (see in Materials and Methods). After growth in galactose for 12 h, the localization of L25-GFP was determined in the fluorescence microscope (A–F). GAL-LOS1ΔN cells were also fixed in 3.7% formaldehyde for 30 min before spheroplasting and indirect immunofluorescence microscopy (G). The L25-GFP signal (fluorescein channel; indicated by arrows), the Nop1p signal (rhodamin channel) and the DNA signal (Hoechst channel) were recorded in a Zeiss Axioskop fluorescence microscope. L25-GFP and Nop1p colocation is indicated by arrows.

Mentions: Since overexpression of a dominant-negative yeast Ran mutant that is stabilized in its GTP-bound form inhibits nucleocytoplasmic transport (Schlenstedt et al., 1995), we tested how this allele affects L25-GFP import and export reactions. Strain L25-GFP was transformed with a plasmid that expresses dominant-negative Gsp1p-G21V under the control of a galactose-inducible promoter. Transformed cells were first grown in repressive conditions, i.e., raffinose-containing medium before shifting them to galactose-containing medium to induce the expression of Gsp1p-G21V. Under repressed conditions, L25-GFP shows an exclusive cytoplasmic location; however, after induction of Gsp1p-G21V, a significant number of cells exhibit nuclear accumulation of L25-GFP that can be distributed throughout the entire nucleus or restricted to a distinct nuclear spot (Fig. 5, compare A with 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)

Accumulation of  L25-GFP inside the nucleus  in strains overexpressing  Los1p and Gsp1p-G21V.  L25-GFP cells carrying the  plasmids (A) pEMBLyex4  (GAL), (B) pGAL-GSP1-G21V, (C) pGAL-KAP95,  (D) pGAL-YRB4ΔN,  (E) pEMBLyex4-ProtA-LOS1 (GAL-LOS1), (F  and G) pEMBLyex4-ProtA-LOS1ΔN (GAL-LOS1ΔN)  were first grown in raffinose-containing medium before  transferring them into galactose-containing medium/ plate (see in Materials and  Methods). After growth in  galactose for 12 h, the localization of L25-GFP was determined in the fluorescence  microscope (A–F). GAL-LOS1ΔN cells were also  fixed in 3.7% formaldehyde  for 30 min before spheroplasting and indirect immunofluorescence microscopy (G).  The L25-GFP signal (fluorescein channel; indicated by arrows), the Nop1p signal  (rhodamin channel) and the  DNA signal (Hoechst channel) were recorded in a Zeiss  Axioskop fluorescence microscope. L25-GFP and  Nop1p colocation is indicated by arrows.
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Figure 5: Accumulation of L25-GFP inside the nucleus in strains overexpressing Los1p and Gsp1p-G21V. L25-GFP cells carrying the plasmids (A) pEMBLyex4 (GAL), (B) pGAL-GSP1-G21V, (C) pGAL-KAP95, (D) pGAL-YRB4ΔN, (E) pEMBLyex4-ProtA-LOS1 (GAL-LOS1), (F and G) pEMBLyex4-ProtA-LOS1ΔN (GAL-LOS1ΔN) were first grown in raffinose-containing medium before transferring them into galactose-containing medium/ plate (see in Materials and Methods). After growth in galactose for 12 h, the localization of L25-GFP was determined in the fluorescence microscope (A–F). GAL-LOS1ΔN cells were also fixed in 3.7% formaldehyde for 30 min before spheroplasting and indirect immunofluorescence microscopy (G). The L25-GFP signal (fluorescein channel; indicated by arrows), the Nop1p signal (rhodamin channel) and the DNA signal (Hoechst channel) were recorded in a Zeiss Axioskop fluorescence microscope. L25-GFP and Nop1p colocation is indicated by arrows.
Mentions: Since overexpression of a dominant-negative yeast Ran mutant that is stabilized in its GTP-bound form inhibits nucleocytoplasmic transport (Schlenstedt et al., 1995), we tested how this allele affects L25-GFP import and export reactions. Strain L25-GFP was transformed with a plasmid that expresses dominant-negative Gsp1p-G21V under the control of a galactose-inducible promoter. Transformed cells were first grown in repressive conditions, i.e., raffinose-containing medium before shifting them to galactose-containing medium to induce the expression of Gsp1p-G21V. Under repressed conditions, L25-GFP shows an exclusive cytoplasmic location; however, after induction of Gsp1p-G21V, a significant number of cells exhibit nuclear accumulation of L25-GFP that can be distributed throughout the entire nucleus or restricted to a distinct nuclear spot (Fig. 5, compare A with 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