Journal article
Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases
Traffic, Vol.18(7), pp.465-484
06/01/2017
DOI: 10.1111/tra.12485
PMCID: PMC5503471
PMID: 28382714
Abstract
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.
Details
- Title: Subtitle
- Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases
- Creators
- Chris MacDonaldStanley Winistorfer - University of Iowa, Molecular Physiology and BiophysicsRobert M Pope - University of Iowa, Medicine AdministrationMichael E Wright - University of Iowa, Molecular Physiology and BiophysicsRobert C Piper - University of Iowa, Medicine Administration
- Resource Type
- Journal article
- Publication Details
- Traffic, Vol.18(7), pp.465-484
- Publisher
- Wiley Subscription Services, Inc; Malden
- DOI
- 10.1111/tra.12485
- PMID
- 28382714
- PMCID
- PMC5503471
- ISSN
- 1398-9219
- eISSN
- 1600-0854
- Grant note
- NIH, Grant/Award numbers: 5R01GM058202, R01GM094830; AHA postdoctoral fellowship award, Grant/Award number: 15POST22980010; Carver Foundation; Thermo Lumos.
- Language
- English
- Date published
- 06/01/2017
- Academic Unit
- Molecular Physiology and Biophysics
- Record Identifier
- 9983757057802771
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