Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases

Traffic. 2017 Jul;18(7):465-484. doi: 10.1111/tra.12485. Epub 2017 Jun 1.


The covalent attachment of ubiquitin onto proteins can elicit a variety of downstream consequences. Attachment is mediated by a large array of E3 ubiquitin ligases, each thought be subject to regulatory control and to have a specific repertoire of substrates. Assessing the biological roles of ligases, and in particular, identifying their biologically relevant substrates has been a persistent yet challenging question. In this study, we describe tools that may help achieve both of these goals. We describe a strategy whereby the activity of a ubiquitin ligase has been enzymatically reversed, accomplished by fusing it to a catalytic domain of an exogenous deubiquitinating enzyme. We present a library of 72 "anti-ligases" that appear to work in a dominant-negative fashion to stabilize their cognate substrates against ubiquitin-dependent proteasomal and lysosomal degradation. We then used the ligase-deubiquitinating enzyme (DUb) library to screen for E3 ligases involved in post-Golgi/endosomal trafficking. We identify ligases previously implicated in these pathways (Rsp5 and Tul1), in addition to ligases previously localized to endosomes (Pib1 and Vps8). We also document an optimized workflow for isolating and analyzing the "ubiquitome" of yeast, which can be used with mass spectrometry to identify substrates perturbed by expression of particular ligase-DUb fusions.

Keywords: E3 ubiquitin ligase; ER-associated degradation (ERAD); endosomal trafficking; lysosome; mass spectrometry; proteasome; ubiquitin; ubiquitination; vacuolar protein sorting.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Membrane / metabolism
  • Deubiquitinating Enzymes / genetics
  • Deubiquitinating Enzymes / metabolism*
  • Endoplasmic Reticulum-Associated Degradation*
  • Endosomal Sorting Complexes Required for Transport / metabolism
  • Endosomes / enzymology
  • Golgi Apparatus / enzymology
  • Lysosomes / enzymology
  • Plasmids
  • Protein Transport
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination


  • Endosomal Sorting Complexes Required for Transport
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • Ubiquitin-Protein Ligases
  • Deubiquitinating Enzymes