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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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Related in: MedlinePlus

Western analysis of 2D PAGE of S. pombe proteins.A. 50 µg of a wild-type total cell extract was separated by IEF (pH 3–6) followed by SDS-PAGE (12.5%) and Western blotted with anti-SUMO antisera. Boxed region is an expanded version of a longer exposure of the same blot. B. Comparison of species in extracts from wt, pmt3-GG and wt extracts+CIP (5 units/50 µg protein). * presumed forms of SUMO monomer, A,B, forms not observed after CIP treatment , # possible acetylated form.
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pone-0006750-g001: Western analysis of 2D PAGE of S. pombe proteins.A. 50 µg of a wild-type total cell extract was separated by IEF (pH 3–6) followed by SDS-PAGE (12.5%) and Western blotted with anti-SUMO antisera. Boxed region is an expanded version of a longer exposure of the same blot. B. Comparison of species in extracts from wt, pmt3-GG and wt extracts+CIP (5 units/50 µg protein). * presumed forms of SUMO monomer, A,B, forms not observed after CIP treatment , # possible acetylated form.

Mentions: As part of our investigation into sumoylated species in S. pombe we undertook 2D PAGE. An example of a typical gel (using a low level of protein, 50 µg, and isoelectric focussing range pH 3–6) stained with colloidal Coomassie Blue is shown in Figure S2. Western blotting of a similar 2D gel with anti-SUMO antisera (Figure 1A) showed multiple species, including five which migrate with pIs and Mr similar to that of SUMO/Pmt3. (S. pombe SUMO/Pmt3 has a predicted pI of 4.6 and migrates at approximately 18 kDa, e.g. [28]).


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)

Western analysis of 2D PAGE of S. pombe proteins.A. 50 µg of a wild-type total cell extract was separated by IEF (pH 3–6) followed by SDS-PAGE (12.5%) and Western blotted with anti-SUMO antisera. Boxed region is an expanded version of a longer exposure of the same blot. B. Comparison of species in extracts from wt, pmt3-GG and wt extracts+CIP (5 units/50 µg protein). * presumed forms of SUMO monomer, A,B, forms not observed after CIP treatment , # possible acetylated form.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006750-g001: Western analysis of 2D PAGE of S. pombe proteins.A. 50 µg of a wild-type total cell extract was separated by IEF (pH 3–6) followed by SDS-PAGE (12.5%) and Western blotted with anti-SUMO antisera. Boxed region is an expanded version of a longer exposure of the same blot. B. Comparison of species in extracts from wt, pmt3-GG and wt extracts+CIP (5 units/50 µg protein). * presumed forms of SUMO monomer, A,B, forms not observed after CIP treatment , # possible acetylated form.
Mentions: As part of our investigation into sumoylated species in S. pombe we undertook 2D PAGE. An example of a typical gel (using a low level of protein, 50 µg, and isoelectric focussing range pH 3–6) stained with colloidal Coomassie Blue is shown in Figure S2. Western blotting of a similar 2D gel with anti-SUMO antisera (Figure 1A) showed multiple species, including five which migrate with pIs and Mr similar to that of SUMO/Pmt3. (S. pombe SUMO/Pmt3 has a predicted pI of 4.6 and migrates at approximately 18 kDa, e.g. [28]).

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