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

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

doi:10.1016/j.xpro.2023.102659

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

STAR Protoc. 2023 Dec 15;4(4):102659. doi: 10.1016/j.xpro.2023.102659. Epub 2023 Oct 26.

Authors

Cameron Williams¹, Ken C Dong², Connor Arkinson², Andreas Martin³

Affiliations

¹ Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA.
² 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.
³ 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. Electronic address: a.martin@berkeley.edu.

Abstract

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.

Keywords: Biophysics; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry.

MeSH terms

Fluorescence Resonance Energy Transfer
Nucleocytoplasmic Transport Proteins / chemistry
Nucleocytoplasmic Transport Proteins / metabolism
Polyubiquitin*
Saccharomyces cerevisiae Proteins* / metabolism
Ubiquitin / metabolism
Valosin Containing Protein / chemistry
Valosin Containing Protein / metabolism

Substances

Polyubiquitin
Saccharomyces cerevisiae Proteins
Valosin Containing Protein
Nucleocytoplasmic Transport Proteins
Ubiquitin

Grants and funding

R01 GM094497/GM/NIGMS NIH HHS/United States