Abstract
Posttranslational modification by the ubiquitin or SUMO (small ubiquitin-like modifier)
polypeptides represents essential as well as evolutionary conserved ways of regulating the
proteomes of eukaryotic cells. Both modifiers generally change the function of their targets by altering their conformation or interactions with other macromolecules. Though, whereas the downstream consequence of ubiquitin conjugation is often protein degradation, the functional outcomes of sumoylation are less unifiable. A class of ubiquitin E3 ligases able to target sumoylated proteins for degradation by the 26S proteasome mediates direct cross-talk between the two modification systems. By contributing to the dynamic turnover of SUMO conjugated species these SUMO-targeted ubiquitin ligases (STUbLs) fulfills essential roles in both yeast and man. However, the specific sumoylated proteins affected by STUbL activity and the specific molecular interactions and sequence of events linking sumoylation, ubiquitylation and substrate degradation, has been largely uncovered.
Using the fission yeast model organism I here present evidence for a role of the Ufd1 (ubiquitinfusion degradation 1) protein, and by extension of the Cdc48-Ufd1-Npl4 complex, in the STUbL pathway. Cdc48-Ufd1-Npl4 forms a highly conserved molecular chaperone. By coupling ubiquitinselective binding with Cdc48 ATPase activity, Cdc48-Ufd1-Npl4 enables the mobilization of ubiquitylated proteins from higher order complexes to promote their degradation or potential other downstream fates. My work provides insight into how Cdc48-Ufd1-Npl4 also contributes to the processing of SUMO conjugates and suggests that at least some of these activities are coordinated with STUbL function. To gain insight into the sumoylated species regulated by Ufd1 and/or by
STUbLs, I made use of a newly developed two-step purification strategy for isolating sumoylated species which allow their site-specific identification by mass spectrometry. In combination with SILAC-based quantitative proteomics I compared sumoylation levels between wild type cells and mutant strains deficient either in STUbL or Ufd1 function. In addition to identifying more than 900 unique sumoylated sites, these efforts revealed a number of proteins with upregulated sumoylation either in STUbL and/or Ufd1 mutant cells. These findings propose specific candidate substrates through which STUbL and Cdc48-Ufd1-Npl4 activities are coupled to dynamically regulate cellular processes.
polypeptides represents essential as well as evolutionary conserved ways of regulating the
proteomes of eukaryotic cells. Both modifiers generally change the function of their targets by altering their conformation or interactions with other macromolecules. Though, whereas the downstream consequence of ubiquitin conjugation is often protein degradation, the functional outcomes of sumoylation are less unifiable. A class of ubiquitin E3 ligases able to target sumoylated proteins for degradation by the 26S proteasome mediates direct cross-talk between the two modification systems. By contributing to the dynamic turnover of SUMO conjugated species these SUMO-targeted ubiquitin ligases (STUbLs) fulfills essential roles in both yeast and man. However, the specific sumoylated proteins affected by STUbL activity and the specific molecular interactions and sequence of events linking sumoylation, ubiquitylation and substrate degradation, has been largely uncovered.
Using the fission yeast model organism I here present evidence for a role of the Ufd1 (ubiquitinfusion degradation 1) protein, and by extension of the Cdc48-Ufd1-Npl4 complex, in the STUbL pathway. Cdc48-Ufd1-Npl4 forms a highly conserved molecular chaperone. By coupling ubiquitinselective binding with Cdc48 ATPase activity, Cdc48-Ufd1-Npl4 enables the mobilization of ubiquitylated proteins from higher order complexes to promote their degradation or potential other downstream fates. My work provides insight into how Cdc48-Ufd1-Npl4 also contributes to the processing of SUMO conjugates and suggests that at least some of these activities are coordinated with STUbL function. To gain insight into the sumoylated species regulated by Ufd1 and/or by
STUbLs, I made use of a newly developed two-step purification strategy for isolating sumoylated species which allow their site-specific identification by mass spectrometry. In combination with SILAC-based quantitative proteomics I compared sumoylation levels between wild type cells and mutant strains deficient either in STUbL or Ufd1 function. In addition to identifying more than 900 unique sumoylated sites, these efforts revealed a number of proteins with upregulated sumoylation either in STUbL and/or Ufd1 mutant cells. These findings propose specific candidate substrates through which STUbL and Cdc48-Ufd1-Npl4 activities are coupled to dynamically regulate cellular processes.
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Number of pages | 194 |
| Publication status | Published - 2014 |