title: Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway.
abstract: The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10delta) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimdelta)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10delta cells, whereas only one-fifth as many accumulated in uimdelta cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS.
authors.affiliation: Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. mayor@interchange.ubc.ca
authors.forename: Thibault
authors.initials: T
authors.lastname: Mayor
authors.forename: Johannes
authors.initials: J
authors.lastname: Graumann
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bryan
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D002352:Carrier Proteins / Q000502:physiology*; D007553:Isotope Labeling; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000502:physiology; D013379:Substrate Specificity; D025801:Ubiquitin / Q000032:analysis*
chemical_list: D002352:Carrier Proteins; D020543:Proteome; C122019:RPN10 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex