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

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

Author information

1
Department of Biochemistry and Biophysics, Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158, USA.
2
Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zürich CH-8093, Switzerland.
3
Department of Biochemistry and Biophysics, Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158, USA. Electronic address: toczyski@cc.ucsf.edu.

Abstract

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.

PMID:
24389104
PMCID:
PMC4032118
DOI:
10.1016/j.molcel.2013.12.003
[Indexed for MEDLINE]
Free PMC Article

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