Quantitative Profiling of Ubiquitylated Proteins Reveals Proteasome Substrates and the Substrate Repertoire Influenced by the Rpn10 Receptor Pathway

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 (rpn10Δ) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimΔ)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10Δ cells, whereas only one-fifth as many accumulated in uimΔ 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. 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 (rpn10Δ) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimΔ)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10Δ cells, whereas only one-fifth as many accumulated in uimΔ 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. The classical function of ubiquitylation is to direct substrates for proteolysis via the ubiquitin proteasome system (UPS). 1The abbreviations used are: UPS, ubiquitin proteasome system; VWA, von Willebrand A; UIM, ubiquitin-interacting motif; FDR, false discovery rate; TAP, tandem affinity purification; MudPIT, multidimensional protein identification technology; WT, wild-type; DUB, deubiquitylating. Recognition of proteasome substrates is specifically mediated by several receptor proteins (1Madura K. Rad23 and Rpn10: perennial wallflowers join the melee.Trends Biochem. Sci. 2004; 29: 637-640Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In yeast, there are at least five potential receptors (Ddi1, Dsk2, Rad23, Rpn10, and Rpt5) plus a set of Cdc48-based complexes, including the Cdc48-Npl4-Ufd1 heterotrimer, that may possess receptor function (2Elsasser S. Chandler-Militello D. Muller B. Hanna J. Finley D. Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome.J. Biol. Chem. 2004; 279: 26817-26822Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 3Ivantsiv Y. Kaplun L. Tzirkin-Goldin R. Shabek N. Raveh D. Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes.Mol. Cell. Biol. 2006; 26: 1579-1588Crossref PubMed Scopus (51) Google Scholar, 4Medicherla B. Kostova Z. Schaefer A. Wolf D.H. A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation.EMBO 2004; PubMed Scopus Google Scholar, R. R. J. receptors a of in the 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A the PubMed Scopus Google Scholar, R. K. 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