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

Mayor, Thibault and Graumann, Johannes and Bryan, Jennnifer and MacCoss, Michael J. and Deshaies, Raymond J. (2007) Quantitative Profiling of Ubiquitylated Proteins Reveals Proteasome Substrates and the Substrate Repertoire Influenced by the Rpn10 Receptor Pathway. Molecular and Cellular Proteomics, 6 (11). pp. 1885-1895. ISSN 1535-9476. https://resolver.caltech.edu/CaltechAUTHORS:MAYmcp07

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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 (rpn10{Delta}) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uim{Delta})). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10{Delta} cells, whereas only one-fifth as many accumulated in uim{Delta} 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1074/mcp.M700264-MCP200DOIArticle
http://www.mcponline.org/content/6/11/1885.longPublisherArticle
ORCID:
AuthorORCID
Deshaies, Raymond J.0000-0002-3671-9354
Additional Information:© 2007 The American Society for Biochemistry and Molecular Biology, Inc. Received, June 5, 2007. Published, MCP Papers in Press, July 20, 2007, DOI 10.1074/mcp.M700264-MCP200. We thank Brian Williams for help in the list analysis and Barbara J. Wold, Rati Verma, Robert Riley, Sonja Hess, Allan D. Drummond, and Kenneth McCue for discussion. We thank all current and past members of the Deshaies laboratory for help, in particular Geoff T. Smith for dedicated technical assistance. T.M. also thanks Gary Kleiger for discussions, encouragement, and shared coffee breaks. This work was supported in part by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The online version of this article (available at http://www.mcponline.org) contains supplemental material. [J.B. was] [s]upported by Natural Sciences and Engineering Research Council (Canada) and the Michael Smith Foundation for Health Research. [M.J.M. was] [s]upported by National Institutes of Health Grant P41-RR011823. [R.J.D. is] an Investigator of the Howard Hughes Medical Institute.
Funders:
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Natural Sciences and Engineering Research Council of Canada (NSERC)UNSPECIFIED
Michael Smith Foundation for Health Research (MSFHR)UNSPECIFIED
NIHP41-RR011823
Issue or Number:11
Record Number:CaltechAUTHORS:MAYmcp07
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:MAYmcp07
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9252
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:01 Dec 2007
Last Modified:12 Dec 2019 17:07

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