title: Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases.
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.
authors.affiliation: Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa.
authors.forename: Chris
authors.initials: C
authors.lastname: MacDonald
authors.affiliation: Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa.
authors.forename: Stanley
authors.initials: S
authors.lastname: Winistorfer
authors.affiliation: Proteomics Facility, University of Iowa, Iowa City, Iowa.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Pope
authors.affiliation: Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Wright
authors.affiliation: Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Piper
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D000072017:Deubiquitinating Enzymes / Q000235:genetics / Q000378:metabolism*; D060746:Endoplasmic Reticulum-Associated Degradation; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D011992:Endosomes / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology; D010957:Plasmids; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000072017:Deubiquitinating Enzymes
keywords: E3 ubiquitin ligase; ER-associated degradation (ERAD); endosomal trafficking; lysosome; mass spectrometry; proteasome; ubiquitin; ubiquitination; vacuolar protein sorting