Limits...
The essential functions of NEDD8 are mediated via distinct surface regions, and not by polyneddylation in Schizosaccharomyces pombe.

Girdwood D, Xirodimas DP, Gordon C - PLoS ONE (2011)

Bottom Line: NEDD8 conjugation to cullin proteins is a prerequisite for cullin based E3 ubiquitin ligase activity, and essential for S. pombe viability.However, we further identified essential residues not previously reported to be involved in ubiquitin ligase regulation that importantly do not prevent Ned8p conjugation.We also find that mutation of all conserved lysine residues in Ned8p, did not affect yeast viability, suggesting that mono-neddylation is sufficient for yeast viability under most conditions.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom. davidgirdwood@gmail.com

ABSTRACT
The ubiquitin-like protein NEDD8 is highly conserved in eukaryotes, from man to Schizosaccharomyces pombe. NEDD8 conjugation to cullin proteins is a prerequisite for cullin based E3 ubiquitin ligase activity, and essential for S. pombe viability. Here, we have performed alanine scanning mutagenesis of all conserved surface residues and show that the majority of essential residues were located around the hydrophobic patch and the C-terminus. However, we further identified essential residues not previously reported to be involved in ubiquitin ligase regulation that importantly do not prevent Ned8p conjugation. We also find that mutation of all conserved lysine residues in Ned8p, did not affect yeast viability, suggesting that mono-neddylation is sufficient for yeast viability under most conditions.

Show MeSH

Related in: MedlinePlus

Conjugation of mutant Nedp constructs.A. Space-filling representation of residues on NEDD8 known to be involved in protein-protein interactions. Residues involved in reported protein-protein interactions are indicated in light blue for non-essential and dark blue for essential. Essential residues not known to be involved in interactions are coloured red. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD). B. S. pombe strains expressing pDUAL-HFF1c-Ned8GG, and indicated mutants. Total cell lysates (108 cells) were prepared under denaturing conditions, and 6 µl of each extract were separated on a 10% SDS-PAGE gel, prior to the blot being probed with anti-FLAG monoclonal antibody. *denotes non-specific band. C. H1299 cells were transfected His6-NEDD8 wild-type and mutants along with HA-Ubc12C111S, Flag-L11, p53 and Myc-Cullin 4 as indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105002&req=5

pone-0020089-g002: Conjugation of mutant Nedp constructs.A. Space-filling representation of residues on NEDD8 known to be involved in protein-protein interactions. Residues involved in reported protein-protein interactions are indicated in light blue for non-essential and dark blue for essential. Essential residues not known to be involved in interactions are coloured red. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD). B. S. pombe strains expressing pDUAL-HFF1c-Ned8GG, and indicated mutants. Total cell lysates (108 cells) were prepared under denaturing conditions, and 6 µl of each extract were separated on a 10% SDS-PAGE gel, prior to the blot being probed with anti-FLAG monoclonal antibody. *denotes non-specific band. C. H1299 cells were transfected His6-NEDD8 wild-type and mutants along with HA-Ubc12C111S, Flag-L11, p53 and Myc-Cullin 4 as indicated.

Mentions: NEDD8 is highly conserved in eukaryotes, with ∼80% identity between that of the H. sapiens and S. pombe orthologues. To identify surface and solvent accessible residues (as identified from the three dimensional structure [28]) required for viability, we performed comprehensive alanine scaning mutagenesis of these conserved and semi-conserved residues (Fig. 1a). Plasmids encoding mutant constructs of ned8 were integrated into a strain carrying an episomal plasmid (ura4) containing wild type ned8+ and deleted for endogenous ned8+. Selection against the version by 5-FOA allowed us to identify which of the Ned8p mutants are sufficient for viability, as assayed for growth at 25°C (Fig. 1b). Viable Ned8p mutants were then further subjected to temperature stress at 36°C and cold sensitivity at 20°C. Of the forty-one evolutionary conserved and semi-conserved surface residues fourteen were essential, and none displayed conditional growth phenotypes. To date, the only known protein-protein interactions with NEDD8 are through its interactions with its cognate conjugation and deconjugation enzymes [29], [30], [31], and the ubiquitin E2, UBC4 [11]. These known sites of contacts were mapped onto a three-dimensional structure of NEDD8 [28]: and indicated in blue. Following discrimination of the residues found to be non-essential (light blue) and essential (dark blue) for these contacts are indicated (Fig. 2a). Novel essential residues, not known to be required for conjugation are indicated in red (Fig. 2a). To address whether the loss of viability was due to the inability of the mutant Ned8p to conjugate, selected mutants were remade in N-terminally tagged vectors and transformed into wild type S. pombe. The strains carrying the L2A, D18A, H68A mutations were all capable of forming, albeit somewhat reduced, higher molecular weight adducts, although no conjugation was detectable for Ile44 or Tyr45 mutants (Fig. 2b). This lack of conjugation could be explained by the stability of the proteins in vivo as no unconjugated Ned8p of the mutants were detected.


The essential functions of NEDD8 are mediated via distinct surface regions, and not by polyneddylation in Schizosaccharomyces pombe.

Girdwood D, Xirodimas DP, Gordon C - PLoS ONE (2011)

Conjugation of mutant Nedp constructs.A. Space-filling representation of residues on NEDD8 known to be involved in protein-protein interactions. Residues involved in reported protein-protein interactions are indicated in light blue for non-essential and dark blue for essential. Essential residues not known to be involved in interactions are coloured red. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD). B. S. pombe strains expressing pDUAL-HFF1c-Ned8GG, and indicated mutants. Total cell lysates (108 cells) were prepared under denaturing conditions, and 6 µl of each extract were separated on a 10% SDS-PAGE gel, prior to the blot being probed with anti-FLAG monoclonal antibody. *denotes non-specific band. C. H1299 cells were transfected His6-NEDD8 wild-type and mutants along with HA-Ubc12C111S, Flag-L11, p53 and Myc-Cullin 4 as indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105002&req=5

pone-0020089-g002: Conjugation of mutant Nedp constructs.A. Space-filling representation of residues on NEDD8 known to be involved in protein-protein interactions. Residues involved in reported protein-protein interactions are indicated in light blue for non-essential and dark blue for essential. Essential residues not known to be involved in interactions are coloured red. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD). B. S. pombe strains expressing pDUAL-HFF1c-Ned8GG, and indicated mutants. Total cell lysates (108 cells) were prepared under denaturing conditions, and 6 µl of each extract were separated on a 10% SDS-PAGE gel, prior to the blot being probed with anti-FLAG monoclonal antibody. *denotes non-specific band. C. H1299 cells were transfected His6-NEDD8 wild-type and mutants along with HA-Ubc12C111S, Flag-L11, p53 and Myc-Cullin 4 as indicated.
Mentions: NEDD8 is highly conserved in eukaryotes, with ∼80% identity between that of the H. sapiens and S. pombe orthologues. To identify surface and solvent accessible residues (as identified from the three dimensional structure [28]) required for viability, we performed comprehensive alanine scaning mutagenesis of these conserved and semi-conserved residues (Fig. 1a). Plasmids encoding mutant constructs of ned8 were integrated into a strain carrying an episomal plasmid (ura4) containing wild type ned8+ and deleted for endogenous ned8+. Selection against the version by 5-FOA allowed us to identify which of the Ned8p mutants are sufficient for viability, as assayed for growth at 25°C (Fig. 1b). Viable Ned8p mutants were then further subjected to temperature stress at 36°C and cold sensitivity at 20°C. Of the forty-one evolutionary conserved and semi-conserved surface residues fourteen were essential, and none displayed conditional growth phenotypes. To date, the only known protein-protein interactions with NEDD8 are through its interactions with its cognate conjugation and deconjugation enzymes [29], [30], [31], and the ubiquitin E2, UBC4 [11]. These known sites of contacts were mapped onto a three-dimensional structure of NEDD8 [28]: and indicated in blue. Following discrimination of the residues found to be non-essential (light blue) and essential (dark blue) for these contacts are indicated (Fig. 2a). Novel essential residues, not known to be required for conjugation are indicated in red (Fig. 2a). To address whether the loss of viability was due to the inability of the mutant Ned8p to conjugate, selected mutants were remade in N-terminally tagged vectors and transformed into wild type S. pombe. The strains carrying the L2A, D18A, H68A mutations were all capable of forming, albeit somewhat reduced, higher molecular weight adducts, although no conjugation was detectable for Ile44 or Tyr45 mutants (Fig. 2b). This lack of conjugation could be explained by the stability of the proteins in vivo as no unconjugated Ned8p of the mutants were detected.

Bottom Line: NEDD8 conjugation to cullin proteins is a prerequisite for cullin based E3 ubiquitin ligase activity, and essential for S. pombe viability.However, we further identified essential residues not previously reported to be involved in ubiquitin ligase regulation that importantly do not prevent Ned8p conjugation.We also find that mutation of all conserved lysine residues in Ned8p, did not affect yeast viability, suggesting that mono-neddylation is sufficient for yeast viability under most conditions.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom. davidgirdwood@gmail.com

ABSTRACT
The ubiquitin-like protein NEDD8 is highly conserved in eukaryotes, from man to Schizosaccharomyces pombe. NEDD8 conjugation to cullin proteins is a prerequisite for cullin based E3 ubiquitin ligase activity, and essential for S. pombe viability. Here, we have performed alanine scanning mutagenesis of all conserved surface residues and show that the majority of essential residues were located around the hydrophobic patch and the C-terminus. However, we further identified essential residues not previously reported to be involved in ubiquitin ligase regulation that importantly do not prevent Ned8p conjugation. We also find that mutation of all conserved lysine residues in Ned8p, did not affect yeast viability, suggesting that mono-neddylation is sufficient for yeast viability under most conditions.

Show MeSH
Related in: MedlinePlus