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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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Assembly of L25-GFP into 60S ribosomes. (A) Western blot analysis of whole cell extracts. 1, CHRS 52 strain; 2,  CHRS 52 strain transformed with pRS314-L25-GFP (single copy  plasmid); 3, CHRS 52 strain transformed with pYEplac112-L25-GFP (high copy plasmid). Equal amounts of cells were loaded on  the SDS–polyacrylamide gel that was blotted onto nitrocellulose  and probed with anti-GFP antibodies. The position of L25-GFP  is shown. (B) Fluorescence microscopy of yeast cells (CHRS 1d)  expressing L25-GFP. The vacuole (V) and nuclear compartment  (N) is indicated in one of the L25-GFP-expressing cells. (C) Ribosome isolation by sucrose gradient centrifugation. Ribosomal  proteins of the 60S and 40S subunits, which were obtained from  CHRS 1d cells expressing L25-GFP, were separated by SDS-PAGE and visualized, respectively, by Coomassie-staining (upper panel) and Western blotting using anti-GFP antibodies  (lower panel). L, load, i.e., whole cell extract of yeast strain  CHRS 1d expressing L25-GFP; M, 10 kD protein ladder standard  (the prominent band corresponds to 50 kD); 1–7, fractions from  the sucrose gradient. The position of the 60S and 40S fractions,  and of the soluble proteins, are indicated.
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Figure 1: Assembly of L25-GFP into 60S ribosomes. (A) Western blot analysis of whole cell extracts. 1, CHRS 52 strain; 2, CHRS 52 strain transformed with pRS314-L25-GFP (single copy plasmid); 3, CHRS 52 strain transformed with pYEplac112-L25-GFP (high copy plasmid). Equal amounts of cells were loaded on the SDS–polyacrylamide gel that was blotted onto nitrocellulose and probed with anti-GFP antibodies. The position of L25-GFP is shown. (B) Fluorescence microscopy of yeast cells (CHRS 1d) expressing L25-GFP. The vacuole (V) and nuclear compartment (N) is indicated in one of the L25-GFP-expressing cells. (C) Ribosome isolation by sucrose gradient centrifugation. Ribosomal proteins of the 60S and 40S subunits, which were obtained from CHRS 1d cells expressing L25-GFP, were separated by SDS-PAGE and visualized, respectively, by Coomassie-staining (upper panel) and Western blotting using anti-GFP antibodies (lower panel). L, load, i.e., whole cell extract of yeast strain CHRS 1d expressing L25-GFP; M, 10 kD protein ladder standard (the prominent band corresponds to 50 kD); 1–7, fractions from the sucrose gradient. The position of the 60S and 40S fractions, and of the soluble proteins, are indicated.

Mentions: L25 was GFP-tagged at its COOH-terminal end and the corresponding L25-GFP fusion construct was inserted into a single or high copy number yeast plasmid. When transformed into a wild-type yeast strain, expression of L25-GFP (calculated molecular mass is 15.6 kD for L25 plus 25 kD for GFP) could be shown by Western analysis using an antibody against the GFP moiety (Fig. 1 A). When yeast cells expressing L25-GFP were inspected in the fluorescence microscope, a cytoplasmic L25-GFP staining with vacuolar and nuclear exclusion was observed (Fig. 1 B). To show assembly of L25-GFP into 60S ribosomal subunits, ribosomes were isolated by sucrose gradient centrifugation from yeast cells that contained authentic L25 but also expressed L25-GFP from a high copy number plasmid. Clearly, L25-GFP is found in the fractions of the sucrose density gradient that contain the 60S large ribosomal subunits, but it is not detected in the adjacent 40S peak fractions (Fig. 1 C). A small portion of L25-GFP was also found on the top of the density gradient where soluble proteins fractionate. This fraction may represent nonassembled L25-GFP.


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)

Assembly of L25-GFP into 60S ribosomes. (A) Western blot analysis of whole cell extracts. 1, CHRS 52 strain; 2,  CHRS 52 strain transformed with pRS314-L25-GFP (single copy  plasmid); 3, CHRS 52 strain transformed with pYEplac112-L25-GFP (high copy plasmid). Equal amounts of cells were loaded on  the SDS–polyacrylamide gel that was blotted onto nitrocellulose  and probed with anti-GFP antibodies. The position of L25-GFP  is shown. (B) Fluorescence microscopy of yeast cells (CHRS 1d)  expressing L25-GFP. The vacuole (V) and nuclear compartment  (N) is indicated in one of the L25-GFP-expressing cells. (C) Ribosome isolation by sucrose gradient centrifugation. Ribosomal  proteins of the 60S and 40S subunits, which were obtained from  CHRS 1d cells expressing L25-GFP, were separated by SDS-PAGE and visualized, respectively, by Coomassie-staining (upper panel) and Western blotting using anti-GFP antibodies  (lower panel). L, load, i.e., whole cell extract of yeast strain  CHRS 1d expressing L25-GFP; M, 10 kD protein ladder standard  (the prominent band corresponds to 50 kD); 1–7, fractions from  the sucrose gradient. The position of the 60S and 40S fractions,  and of the soluble proteins, are indicated.
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Related In: Results  -  Collection

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Figure 1: Assembly of L25-GFP into 60S ribosomes. (A) Western blot analysis of whole cell extracts. 1, CHRS 52 strain; 2, CHRS 52 strain transformed with pRS314-L25-GFP (single copy plasmid); 3, CHRS 52 strain transformed with pYEplac112-L25-GFP (high copy plasmid). Equal amounts of cells were loaded on the SDS–polyacrylamide gel that was blotted onto nitrocellulose and probed with anti-GFP antibodies. The position of L25-GFP is shown. (B) Fluorescence microscopy of yeast cells (CHRS 1d) expressing L25-GFP. The vacuole (V) and nuclear compartment (N) is indicated in one of the L25-GFP-expressing cells. (C) Ribosome isolation by sucrose gradient centrifugation. Ribosomal proteins of the 60S and 40S subunits, which were obtained from CHRS 1d cells expressing L25-GFP, were separated by SDS-PAGE and visualized, respectively, by Coomassie-staining (upper panel) and Western blotting using anti-GFP antibodies (lower panel). L, load, i.e., whole cell extract of yeast strain CHRS 1d expressing L25-GFP; M, 10 kD protein ladder standard (the prominent band corresponds to 50 kD); 1–7, fractions from the sucrose gradient. The position of the 60S and 40S fractions, and of the soluble proteins, are indicated.
Mentions: L25 was GFP-tagged at its COOH-terminal end and the corresponding L25-GFP fusion construct was inserted into a single or high copy number yeast plasmid. When transformed into a wild-type yeast strain, expression of L25-GFP (calculated molecular mass is 15.6 kD for L25 plus 25 kD for GFP) could be shown by Western analysis using an antibody against the GFP moiety (Fig. 1 A). When yeast cells expressing L25-GFP were inspected in the fluorescence microscope, a cytoplasmic L25-GFP staining with vacuolar and nuclear exclusion was observed (Fig. 1 B). To show assembly of L25-GFP into 60S ribosomal subunits, ribosomes were isolated by sucrose gradient centrifugation from yeast cells that contained authentic L25 but also expressed L25-GFP from a high copy number plasmid. Clearly, L25-GFP is found in the fractions of the sucrose density gradient that contain the 60S large ribosomal subunits, but it is not detected in the adjacent 40S peak fractions (Fig. 1 C). A small portion of L25-GFP was also found on the top of the density gradient where soluble proteins fractionate. This fraction may represent nonassembled L25-GFP.

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