Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing

A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we presen...

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Autores principales: Williams, Cameron, Dong, Ken C., Arkinson, Connor, Martin, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10630674/
https://www.ncbi.nlm.nih.gov/pubmed/37889757
http://dx.doi.org/10.1016/j.xpro.2023.102659
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author Williams, Cameron
Dong, Ken C.
Arkinson, Connor
Martin, Andreas
author_facet Williams, Cameron
Dong, Ken C.
Arkinson, Connor
Martin, Andreas
author_sort Williams, Cameron
collection PubMed
description A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. For complete details on the use and execution of this protocol, please refer to Williams et al. (2023).(1)
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spelling pubmed-106306742023-10-26 Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing Williams, Cameron Dong, Ken C. Arkinson, Connor Martin, Andreas STAR Protoc Protocol A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. For complete details on the use and execution of this protocol, please refer to Williams et al. (2023).(1) Elsevier 2023-10-26 /pmc/articles/PMC10630674/ /pubmed/37889757 http://dx.doi.org/10.1016/j.xpro.2023.102659 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Williams, Cameron
Dong, Ken C.
Arkinson, Connor
Martin, Andreas
Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing
title Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing
title_full Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing
title_fullStr Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing
title_full_unstemmed Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing
title_short Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing
title_sort preparation of site-specifically fluorophore-labeled polyubiquitin chains for fret studies of cdc48 substrate processing
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10630674/
https://www.ncbi.nlm.nih.gov/pubmed/37889757
http://dx.doi.org/10.1016/j.xpro.2023.102659
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