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

doi:10.1074/mcp.M700264-MCP200

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

From the ^‡Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, California 91125, ^§Department of Biochemistry and Molecular Biology, UBC Centre for Proteomics and ^‖Department of Statistics, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada, and ^‡‡Department of Genome Sciences, Health Sciences Center, University of Washington, Seattle, Washington 98195

¶To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, UBC Centre for Proteomics, University of British Columbia, 301-2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada. Tel.: 604-822-5144; Fax: 604-822-2114; E-mail: mayor{at}interchange.ubc.ca

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 (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.

Footnotes

Published, MCP Papers in Press, July 20, 2007, DOI 10.1074/mcp.M700264-MCP200
↵¹ The 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.
↵² J. R. Lipford, personal communication.
↵³ A. Dabney and J. D. Story, personal communication.
↵⁴ T. Mayor and R. J. Deshaies, unpublished data.
↵* 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.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
↵** Supported by Natural Sciences and Engineering Research Council (Canada) and the Michael Smith Foundation for Health Research.
↵§§ Supported by National Institutes of Health Grant P41-RR011823.
↵¶¶ An Investigator of the Howard Hughes Medical Institute.
- Received June 5, 2007.