title: Ubiquitin ligase trapping identifies an SCF(Saf1) pathway targeting unprocessed vacuolar/lysosomal proteins.
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.
authors.affiliation: Department of Biochemistry and Biophysics, Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158, USA.
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Mark
authors.affiliation: Department of Biochemistry and Biophysics, Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Simonetta
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zürich CH-8093, Switzerland.
authors.forename: Alessio
authors.initials: A
authors.lastname: Maiolica
authors.affiliation: Department of Biochemistry and Biophysics, Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Seller
authors.affiliation: Department of Biochemistry and Biophysics, Helen Diller Family Comprehensive Cancer Center, San Francisco, CA 94158, USA. Electronic address: toczyski@cc.ucsf.edu.
authors.forename: David P
authors.initials: DP
authors.lastname: Toczyski
mesh_terms: D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism*
chemical_list: D044783:F-Box Proteins; D029701:Saccharomyces cerevisiae Proteins; C512528:Saf1 protein, S cerevisiae; D057149:Ubiquitinated Proteins; D044767:Ubiquitin-Protein Ligases