Ubiquitin ligase trapping identifies an SCF(Saf1) pathway targeting unprocessed vacuolar/lysosomal proteins

Mol Cell. 2014 Jan 9;53(1):148-61. doi: 10.1016/j.molcel.2013.12.003. Epub 2014 Jan 2.


We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin-associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using ligase traps of eight different F box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously unreported, substrates. Polyubiquitinated forms of candidate substrates associated with their cognate F box partner, but not other ligase traps. Interestingly, the four most abundant candidate substrates identified for the F box protein Saf1 were all vacuolar/lysosomal proteins. Analysis of one of these substrates, Prb1, showed that Saf1 selectively promotes ubiquitination of the unprocessed form of the zymogen. This suggests that Saf1 is part of a pathway that targets protein precursors for proteasomal degradation.

Publication types

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

MeSH terms

  • F-Box Proteins / genetics
  • F-Box Proteins / metabolism*
  • Lysosomes / genetics
  • Lysosomes / metabolism*
  • Mass Spectrometry
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitinated Proteins / genetics
  • Ubiquitinated Proteins / metabolism*
  • Ubiquitination / physiology
  • Vacuoles / genetics
  • Vacuoles / metabolism*


  • F-Box Proteins
  • Saccharomyces cerevisiae Proteins
  • Saf1 protein, S cerevisiae
  • Ubiquitinated Proteins
  • Ubiquitin-Protein Ligases