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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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Growth of L25-GFP-expressing strains. (A) L25-GFP  complements a rpl25::HIS3  mutant. (Upper and middle  panel) Tetrad analysis of the heterozygous rpl25::HIS3/RPL25  diploid strain reveals a 2:2 segregation, indicating that the RPL25  gene is essential for viability. Viability of the rpl25::HIS3 spores  is rescued by cosegregation of the pYEplac195-ADE2-URA3-L25-GFP plasmid with chromosomal rpl25::HIS3. (Lower panel)  Growth properties of haploid L25-GFP strain. Precultures were  diluted in growth medium and equivalent amounts of cells (diluted in 10−1 steps) were spotted onto YPD plates. hRS453 is a  haploid wild-type, L25-GFP a haploid strain disrupted for rlp25:: HIS3 and complemented by YEplac195-ADE2-URA3-L25-GFP. It was grown for 4 d. (B–D) Growth properties of different  single and double mutant strains at different temperatures. Precultures were diluted in growth medium and equivalent amounts  of cells (diluted in 10−1 steps) were spotted onto YPD plates.  Cells were grown at the indicated temperatures and for the indicated days on YPD plates. For description of strains, see Materials and Methods and Table I.
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Figure 2: Growth of L25-GFP-expressing strains. (A) L25-GFP complements a rpl25::HIS3 mutant. (Upper and middle panel) Tetrad analysis of the heterozygous rpl25::HIS3/RPL25 diploid strain reveals a 2:2 segregation, indicating that the RPL25 gene is essential for viability. Viability of the rpl25::HIS3 spores is rescued by cosegregation of the pYEplac195-ADE2-URA3-L25-GFP plasmid with chromosomal rpl25::HIS3. (Lower panel) Growth properties of haploid L25-GFP strain. Precultures were diluted in growth medium and equivalent amounts of cells (diluted in 10−1 steps) were spotted onto YPD plates. hRS453 is a haploid wild-type, L25-GFP a haploid strain disrupted for rlp25:: HIS3 and complemented by YEplac195-ADE2-URA3-L25-GFP. It was grown for 4 d. (B–D) Growth properties of different single and double mutant strains at different temperatures. Precultures were diluted in growth medium and equivalent amounts of cells (diluted in 10−1 steps) were spotted onto YPD plates. Cells were grown at the indicated temperatures and for the indicated days on YPD plates. For description of strains, see Materials and Methods and Table I.

Mentions: To find out whether L25-GFP can functionally replace authentic L25, the RPL25 gene that encodes the L25 ribosomal protein was disrupted in a diploid yeast strain. Tetrad analysis of sporulated heterozygous diploids (rpl25::HIS3/ RPL25) revealed a 2:2 segregation for viability (Fig. 2 A). This confirmed that RPL25 is essential for cell growth (Rutgers et al., 1990). The lethal phenotype of haploid rpl25:HIS3 progeny can be complemented by the expression of plasmid-borne L25-GFP, although L25-GFP cells grow slower than wild-type cells (Fig. 2 A). However, L25-GFP cells do not exhibit a particular heat- or cold-sensitive phenotype (Fig. 2 A, compare hRS453 and L25-GFP). This shows that the attachment of GFP to L25 only slightly impairs the L25 function. When haploid cells with disrupted RPL25 gene but complemented by plasmid-borne L25-GFP were inspected in the fluorescence microscope, the GFP signal increased and was still exclusively cytoplasmic (see also below). This suggests that L25-GFP assembles more efficiently into 60S ribosomal subunits when the endogenous L25 copy is not present as a competitor. This is consistent with the observation that no free L25-GFP can be detected in the upper part of a sucrose gradient when a whole cell extract derived from L25-GFP cells was analyzed (see also Fig. 1 C); instead L25-GFP was now found exclusively associated with 60S ribosomes (data not shown). In conclusion, L25 tagged with the GFP is functional in yeast, since it can replace authentic L25; accordingly, L25-GFP carrying ribosomes are exported from the nucleus into the cytoplasm.


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)

Growth of L25-GFP-expressing strains. (A) L25-GFP  complements a rpl25::HIS3  mutant. (Upper and middle  panel) Tetrad analysis of the heterozygous rpl25::HIS3/RPL25  diploid strain reveals a 2:2 segregation, indicating that the RPL25  gene is essential for viability. Viability of the rpl25::HIS3 spores  is rescued by cosegregation of the pYEplac195-ADE2-URA3-L25-GFP plasmid with chromosomal rpl25::HIS3. (Lower panel)  Growth properties of haploid L25-GFP strain. Precultures were  diluted in growth medium and equivalent amounts of cells (diluted in 10−1 steps) were spotted onto YPD plates. hRS453 is a  haploid wild-type, L25-GFP a haploid strain disrupted for rlp25:: HIS3 and complemented by YEplac195-ADE2-URA3-L25-GFP. It was grown for 4 d. (B–D) Growth properties of different  single and double mutant strains at different temperatures. Precultures were diluted in growth medium and equivalent amounts  of cells (diluted in 10−1 steps) were spotted onto YPD plates.  Cells were grown at the indicated temperatures and for the indicated days on YPD plates. For description of strains, see Materials and Methods and Table I.
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Related In: Results  -  Collection

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Figure 2: Growth of L25-GFP-expressing strains. (A) L25-GFP complements a rpl25::HIS3 mutant. (Upper and middle panel) Tetrad analysis of the heterozygous rpl25::HIS3/RPL25 diploid strain reveals a 2:2 segregation, indicating that the RPL25 gene is essential for viability. Viability of the rpl25::HIS3 spores is rescued by cosegregation of the pYEplac195-ADE2-URA3-L25-GFP plasmid with chromosomal rpl25::HIS3. (Lower panel) Growth properties of haploid L25-GFP strain. Precultures were diluted in growth medium and equivalent amounts of cells (diluted in 10−1 steps) were spotted onto YPD plates. hRS453 is a haploid wild-type, L25-GFP a haploid strain disrupted for rlp25:: HIS3 and complemented by YEplac195-ADE2-URA3-L25-GFP. It was grown for 4 d. (B–D) Growth properties of different single and double mutant strains at different temperatures. Precultures were diluted in growth medium and equivalent amounts of cells (diluted in 10−1 steps) were spotted onto YPD plates. Cells were grown at the indicated temperatures and for the indicated days on YPD plates. For description of strains, see Materials and Methods and Table I.
Mentions: To find out whether L25-GFP can functionally replace authentic L25, the RPL25 gene that encodes the L25 ribosomal protein was disrupted in a diploid yeast strain. Tetrad analysis of sporulated heterozygous diploids (rpl25::HIS3/ RPL25) revealed a 2:2 segregation for viability (Fig. 2 A). This confirmed that RPL25 is essential for cell growth (Rutgers et al., 1990). The lethal phenotype of haploid rpl25:HIS3 progeny can be complemented by the expression of plasmid-borne L25-GFP, although L25-GFP cells grow slower than wild-type cells (Fig. 2 A). However, L25-GFP cells do not exhibit a particular heat- or cold-sensitive phenotype (Fig. 2 A, compare hRS453 and L25-GFP). This shows that the attachment of GFP to L25 only slightly impairs the L25 function. When haploid cells with disrupted RPL25 gene but complemented by plasmid-borne L25-GFP were inspected in the fluorescence microscope, the GFP signal increased and was still exclusively cytoplasmic (see also below). This suggests that L25-GFP assembles more efficiently into 60S ribosomal subunits when the endogenous L25 copy is not present as a competitor. This is consistent with the observation that no free L25-GFP can be detected in the upper part of a sucrose gradient when a whole cell extract derived from L25-GFP cells was analyzed (see also Fig. 1 C); instead L25-GFP was now found exclusively associated with 60S ribosomes (data not shown). In conclusion, L25 tagged with the GFP is functional in yeast, since it can replace authentic L25; accordingly, L25-GFP carrying ribosomes are exported from the nucleus into the cytoplasm.

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