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

Cameron Williams; Ken C. Dong; Connor Arkinson; Andreas Martin

STAR Protocols (Dec 2023)

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

Cameron Williams,
Ken C. Dong,
Connor Arkinson,
Andreas Martin

Affiliations

Cameron Williams: Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA
Ken C. Dong: California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California at Berkeley, Berkeley CA 94720
Connor Arkinson: California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California at Berkeley, Berkeley CA 94720
Andreas Martin: California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California at Berkeley, Berkeley CA 94720; Corresponding author

Journal volume & issue: Vol. 4, no. 4
p. 102659

Abstract

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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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