To investigate potential interplay between the SUMO1 (small ubiquitin-related modifier-1) and ubiquitin pathways of post-translational protein modification, we examined aspects of their localization and conjugation status during proteasome inhibition. Our results indicate that these pathways converge upon the discrete sub-nuclear domains known as PML (promyelocytic leukaemia protein) NBs (nuclear bodies). Proteasome inhibition generated an increased number of PML bodies, without any obvious increase in size. Using a cell line that constitutively expresses an epitope-tagged version of SUMO1, which was incorporated into high-molecular-mass conjugates, we observed SUMO1 accumulating in clusters around a subset of the NBs. Nuclear ubiquitin was initially observed in numerous speckles and foci, which bore no relationship to PML NBs in the absence of proteasome inhibition. However, during proteasome inhibition, total ubiquitin-conjugated species increased in the cell, as judged by Western blotting. Concomitantly the number of nuclear ubiquitin clusters decreased, and were almost quantitatively associated with the PML NBs, co-localizing with the SUMO-conjugated pool. Proteasome inhibition depleted the pool of free SUMO1 in the cell. Reversal of proteasome inhibition in the presence or absence of protein synthesis demonstrated that free SUMO1 was regenerated from the conjugated pool. The results indicate that a significant fraction of the free SUMO1 pool could be accounted for by recycling from the conjugated pool and indeed it may be that, as for ubiquitin, SUMO1 needs to be removed from conjugated species prior to processing by the proteasome. Taken together with other recent reports on the proteasome and PML NBs, these results suggest that the PML NBs may play an important role in integrating these pathways.
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December 2005
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Research Article|
November 22 2005
Comparison of the SUMO1 and ubiquitin conjugation pathways during the inhibition of proteasome activity with evidence of SUMO1 recycling
Daniel Bailey;
Daniel Bailey
1Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 OTL, U.K.
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Peter O'Hare
Peter O'Hare
1
1Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 OTL, U.K.
1To whom correspondence should be addressed (email P.OHare@mcri.ac.uk).
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Publisher: Portland Press Ltd
Received:
May 31 2005
Revision Received:
July 21 2005
Accepted:
August 24 2005
Accepted Manuscript online:
August 24 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 392 (2): 271–281.
Article history
Received:
May 31 2005
Revision Received:
July 21 2005
Accepted:
August 24 2005
Accepted Manuscript online:
August 24 2005
Citation
Daniel Bailey, Peter O'Hare; Comparison of the SUMO1 and ubiquitin conjugation pathways during the inhibition of proteasome activity with evidence of SUMO1 recycling. Biochem J 1 December 2005; 392 (2): 271–281. doi: https://doi.org/10.1042/BJ20050873
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