Molecular Cell
Volume 53, Issue 1, 9 January 2014, Pages 148-161
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Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins

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Highlights

  • Fusing ubiquitin binding domains to F box proteins allows capture of SCF substrates

  • A mass spectrometry-based ligase trap screen identified 18 candidate SCF targets

  • SCFSaf1 ubiquitinates the unprocessed zymogen form of vacuolar/lysosomal proteases

Summary

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.

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3

These authors contributed equally to this work

4

Present address: The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands