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Originally published In Press as doi:10.1074/jbc.M211240200 on January 10, 2003

J. Biol. Chem., Vol. 278, Issue 11, 9448-9457, March 14, 2003
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Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway*

Thomas J. Siepmann, Richard N. Bohnsack, Zeynep Tokgöz, Olga V. BaboshinaDagger , and Arthur L. Haas§

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E214kb transthiolation is hyperbolic and yields Km values of 102 ± 13 nM and 123 ± 19 nM for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (Ki = 104 ± 15 nM) and HsUbc2bC88S-ubiquitin oxyester (Ki = 169 ± 17 nM), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha -catalyzed conjugation to human alpha -lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a Ki of 54 ± 18 nM and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (Ki = 66 ± 29 nM). In contrast, the ligase product analog HsUbc2bC88A exhibits a Ki of 440 ± 55 nM with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric 125I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.

* This work was supported by United States Public Health Service Grant GM34009 (to A. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Genetics, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104.

§ To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8768; Fax: 414-456-6510; E-mail: arthaas@mcw.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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