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SUMO chain formation is required for response to replication arrest in S. pombe.

Skilton A, Ho JC, Mercer B, Outwin E, Watts FZ - PLoS ONE (2009)

Bottom Line: Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability.Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress.Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, UK.

ABSTRACT
SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved betabetaalphabetabetaalphabeta fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe.

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Phenotype of mutants defective in SUMO/Pmt3 chain formation.A. Western analysis of total cell extracts from cells containing mutant versions of SUMO/Pmt3. Lane 1,5 wt, lane 2 pmt3-K14R, lane 3 pmt3-K30R, lane 4 pmt3-K14R,K30R, lane 6 pli1-d, lane 7 hus5-62. B. Morphology of methanol fixed cells, stained with DAPI and calcofluor. C. Phenotype of pmt3 mutants. 10 µl of 10 fold serially diluted cultures were plated onto media as indicated, and incubated at 25°C for 5 days.
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pone-0006750-g006: Phenotype of mutants defective in SUMO/Pmt3 chain formation.A. Western analysis of total cell extracts from cells containing mutant versions of SUMO/Pmt3. Lane 1,5 wt, lane 2 pmt3-K14R, lane 3 pmt3-K30R, lane 4 pmt3-K14R,K30R, lane 6 pli1-d, lane 7 hus5-62. B. Morphology of methanol fixed cells, stained with DAPI and calcofluor. C. Phenotype of pmt3 mutants. 10 µl of 10 fold serially diluted cultures were plated onto media as indicated, and incubated at 25°C for 5 days.

Mentions: We were next interested in determining whether any of the pmt3 K to R mutations affect SUMO modification or chain formation when the mutant alleles are present in cells as the sole copy of SUMO/Pmt3 and whether they affect cell viability or morphology. All three mutants are viable, although pmt3-K14R,K30R colonies grow slightly slower than wild type (data not shown). Western blotting with anti-SUMO antisera indicates that, compared to wild type, pmt3-K14R has substantially reduced levels of high Mr SUMO-containing species (Figure 6A, lanes 2), compared to wild type cells (lanes 1,5). Cells containing pmt3-K30R (lane 3) have a similar level of high Mr SUMO/Pmt3-containing species to that observed in wild type cells (lanes 1,5), but the double mutant pmt3-K14R,K30R has significantly reduced levels high Mr species (lane 4), intermediate between those observed in pli1-d and hus5-62 cells (defective in the SUMO conjugator, [24], [25]). These data show that K14 and, to a lesser extent, K30 are required for SUMO chain formation in vivo.


SUMO chain formation is required for response to replication arrest in S. pombe.

Skilton A, Ho JC, Mercer B, Outwin E, Watts FZ - PLoS ONE (2009)

Phenotype of mutants defective in SUMO/Pmt3 chain formation.A. Western analysis of total cell extracts from cells containing mutant versions of SUMO/Pmt3. Lane 1,5 wt, lane 2 pmt3-K14R, lane 3 pmt3-K30R, lane 4 pmt3-K14R,K30R, lane 6 pli1-d, lane 7 hus5-62. B. Morphology of methanol fixed cells, stained with DAPI and calcofluor. C. Phenotype of pmt3 mutants. 10 µl of 10 fold serially diluted cultures were plated onto media as indicated, and incubated at 25°C for 5 days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2727700&req=5

pone-0006750-g006: Phenotype of mutants defective in SUMO/Pmt3 chain formation.A. Western analysis of total cell extracts from cells containing mutant versions of SUMO/Pmt3. Lane 1,5 wt, lane 2 pmt3-K14R, lane 3 pmt3-K30R, lane 4 pmt3-K14R,K30R, lane 6 pli1-d, lane 7 hus5-62. B. Morphology of methanol fixed cells, stained with DAPI and calcofluor. C. Phenotype of pmt3 mutants. 10 µl of 10 fold serially diluted cultures were plated onto media as indicated, and incubated at 25°C for 5 days.
Mentions: We were next interested in determining whether any of the pmt3 K to R mutations affect SUMO modification or chain formation when the mutant alleles are present in cells as the sole copy of SUMO/Pmt3 and whether they affect cell viability or morphology. All three mutants are viable, although pmt3-K14R,K30R colonies grow slightly slower than wild type (data not shown). Western blotting with anti-SUMO antisera indicates that, compared to wild type, pmt3-K14R has substantially reduced levels of high Mr SUMO-containing species (Figure 6A, lanes 2), compared to wild type cells (lanes 1,5). Cells containing pmt3-K30R (lane 3) have a similar level of high Mr SUMO/Pmt3-containing species to that observed in wild type cells (lanes 1,5), but the double mutant pmt3-K14R,K30R has significantly reduced levels high Mr species (lane 4), intermediate between those observed in pli1-d and hus5-62 cells (defective in the SUMO conjugator, [24], [25]). These data show that K14 and, to a lesser extent, K30 are required for SUMO chain formation in vivo.

Bottom Line: Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability.Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress.Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, UK.

ABSTRACT
SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved betabetaalphabetabetaalphabeta fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe.

Show MeSH
Related in: MedlinePlus