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Published May 2007 | Published
Journal Article Open

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome


E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.

Additional Information

Copyright © 2007 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0). Submitted October 30, 2006; Revised February 20, 2007; Accepted March 1, 2007. Originally published as MBC in Press, 10.1091/mbc.E06-10-0965 on March 14, 2007. We thank members of the Deshaies laboratory, especially R. Verma and R. Oania, for reagents and helpful discussions and R. Verma for critical reading of the manuscript. We gratefully acknowledge R. Diamond and P. Koen (Caltech Cell Sorting Facility) for performing flow cytometry and J. Graumann, T. Mayor, and G. Smith (Caltech) for mass spectrometry analysis. We thank A. Varshavsky, R. Hampton, K. Madura, M. Funakoshi, R. Vierstra, L. Johnston, and J. Monaco for generously providing reagents. R.D. is an investigator of the Howard Hughes Medical Institute, and this work was supported by Howard Hughes Medical Institute.

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