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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.

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Essential surface residues of NEDD8 and ubiquitin.A. Alignment of sequences of S. pombe Nedp and ubiquitin. Closed circles (•) above Nedp and below ubiquitin denote the essential surface residues. Black boxes indicate identical residues and grey boxes, conservative substitutions. B. Space-filling representation of essential surface residues on NEDD8 and ubiquitin. Non-essential conserved residues are shown in light red, essential conserved in red, and essential non-conserved residues in orange, and two views of NEDD8 and ubiquitin are shown. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD) and ubiquitin (PDB 1UBQ).
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pone-0020089-g003: Essential surface residues of NEDD8 and ubiquitin.A. Alignment of sequences of S. pombe Nedp and ubiquitin. Closed circles (•) above Nedp and below ubiquitin denote the essential surface residues. Black boxes indicate identical residues and grey boxes, conservative substitutions. B. Space-filling representation of essential surface residues on NEDD8 and ubiquitin. Non-essential conserved residues are shown in light red, essential conserved in red, and essential non-conserved residues in orange, and two views of NEDD8 and ubiquitin are shown. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD) and ubiquitin (PDB 1UBQ).

Mentions: As expected from the high sequence and structural similarities between Ned8p and ubiquitin there was significant overlap in the essential residues identified (Fig. 3a) [18]. Conserved, essential (red) and non-essential residues (light red) were mapped onto three dimensional structures of both NEDD8 and ubiquitin [34] (Fig. 3b). The C-terminal tails of both Ned8p and ubiquitin possess a large number of essential amino acids. Indeed, seven of the sixteen essential ubiquitin surface residues are located in the C-terminal tail [18], compared to seven of NEDD8's fourteen essential residues. In contrast to ubiquitin, the conserved canonical hydrophobic patch on Ned8p was not strictly essential for S. pombe viability, as Leu8 mutants were viable (Fig. 1b) [35]. However, as with ubiquitin, the mutation of Ile44 or Val70 resulted in lethality. As such, both the ubiquitin and Ned8p hydrophobic patches appear to be essential for viability, but the requirement is less strict for Ned8p, as demonstrated by mutation of Leu8. Of the conserved Ned8p residues, which have diverged from ubiquitin, only Leu2, Gly10, Asp18 and Arg25, are essential for viability (Fig. 3a).


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)

Essential surface residues of NEDD8 and ubiquitin.A. Alignment of sequences of S. pombe Nedp and ubiquitin. Closed circles (•) above Nedp and below ubiquitin denote the essential surface residues. Black boxes indicate identical residues and grey boxes, conservative substitutions. B. Space-filling representation of essential surface residues on NEDD8 and ubiquitin. Non-essential conserved residues are shown in light red, essential conserved in red, and essential non-conserved residues in orange, and two views of NEDD8 and ubiquitin are shown. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD) and ubiquitin (PDB 1UBQ).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020089-g003: Essential surface residues of NEDD8 and ubiquitin.A. Alignment of sequences of S. pombe Nedp and ubiquitin. Closed circles (•) above Nedp and below ubiquitin denote the essential surface residues. Black boxes indicate identical residues and grey boxes, conservative substitutions. B. Space-filling representation of essential surface residues on NEDD8 and ubiquitin. Non-essential conserved residues are shown in light red, essential conserved in red, and essential non-conserved residues in orange, and two views of NEDD8 and ubiquitin are shown. Figures were made using MacPyMOL on NEDD8 (PDB 1NDD) and ubiquitin (PDB 1UBQ).
Mentions: As expected from the high sequence and structural similarities between Ned8p and ubiquitin there was significant overlap in the essential residues identified (Fig. 3a) [18]. Conserved, essential (red) and non-essential residues (light red) were mapped onto three dimensional structures of both NEDD8 and ubiquitin [34] (Fig. 3b). The C-terminal tails of both Ned8p and ubiquitin possess a large number of essential amino acids. Indeed, seven of the sixteen essential ubiquitin surface residues are located in the C-terminal tail [18], compared to seven of NEDD8's fourteen essential residues. In contrast to ubiquitin, the conserved canonical hydrophobic patch on Ned8p was not strictly essential for S. pombe viability, as Leu8 mutants were viable (Fig. 1b) [35]. However, as with ubiquitin, the mutation of Ile44 or Val70 resulted in lethality. As such, both the ubiquitin and Ned8p hydrophobic patches appear to be essential for viability, but the requirement is less strict for Ned8p, as demonstrated by mutation of Leu8. Of the conserved Ned8p residues, which have diverged from ubiquitin, only Leu2, Gly10, Asp18 and Arg25, are essential for viability (Fig. 3a).

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