Ub-ProT reveals global length and composition of protein ubiquitylation in cells

Nat Commun. 2018 Feb 6;9(1):524. doi: 10.1038/s41467-018-02869-x.


Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Proteins
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / metabolism*
  • HeLa Cells
  • Humans
  • Leupeptins / pharmacology
  • Polyubiquitin / analysis*
  • Polyubiquitin / chemistry
  • Polyubiquitin / metabolism
  • Proteins / metabolism*
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Ubiquitin / chemistry
  • Ubiquitin / metabolism
  • Ubiquitinated Proteins / metabolism
  • Ubiquitination*
  • Valosin Containing Protein / genetics
  • Valosin Containing Protein / metabolism*


  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Proteins
  • Carrier Proteins
  • Cell Cycle Proteins
  • Leupeptins
  • Proteins
  • Saccharomyces cerevisiae Proteins
  • UBQLN1 protein, human
  • Ubiquitin
  • Ubiquitinated Proteins
  • Polyubiquitin
  • Epidermal Growth Factor
  • EGFR protein, human
  • ErbB Receptors
  • CDC48 protein, S cerevisiae
  • Valosin Containing Protein
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde