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Bivalent Inhibitor of the N-end Rule Pathway

Kwon, Yong Tae and Lévy, Frédéric and Varshavsky, Alexander (1999) Bivalent Inhibitor of the N-end Rule Pathway. Journal of Biological Chemistry, 274 (25). pp. 18135-18139. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:KWOjbc99

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Abstract

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.


Item Type:Article
Additional Information:Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, February 9, 1999) We thank members of the Varshavsky laboratory, especially A. Kashina and G. Turner, for helpful discussions and advice in the course of this work. We also thank I. Davydov, F. Du, F. Navarro-Garcia, H. Rao, and especially G. Turner for comments on the manuscript. This work was supported by National Institutes of Health Grant DK39520 (to A. V.).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.
Record Number:CaltechAUTHORS:KWOjbc99
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:KWOjbc99
Alternative URL:http://www.jbc.org/cgi/content/abstract/274/25/18135
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ID Code:6413
Collection:CaltechAUTHORS
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Deposited On:07 Dec 2006
Last Modified:26 Dec 2012 09:21

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