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Published June 2005 | Published
Journal Article Open

Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets


The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10 cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10) and identified 54 proteins that were uniquely recovered from rpn10 cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely inabundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.

Additional Information

© 2005 The American Society for Biochemistry and Molecular Biology, Inc. Received, December 28, 2004, and in revised form, February 7, 2005; published, MCP Papers in Press, February 8, 2005. We thank K. R. Yamamoto, H. Kobayashi, and H. Yokosawa for providing reagents and B. M. Padhiar for technical assistance. We thank all current and past members of the Deshaies laboratory, and in particular G. Alexandru, G. Kleiger, and R. Verma, for technical advice, helpful discussions, and support. We thank J. R. Yates III for providing Sequest, 2to3, DTASelect, and Contrast. R. J. D. and J. G. thank J. R. Yates III, M. MacCoss, and W. H. MacDonald. Without their support, encouragement, and teaching, this work would not have been possible. R J. D. also thanks R. S. Annan and S. A. Carr for introducing him to the power and possibilities of mass spectrometry.

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