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Published June 5, 1993 | Published
Journal Article Open

N-recognin/Ubc2 interactions in the N-end rule pathway


The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, substrates of the N-end rule pathway are targeted for degradation by a complex that includes the 225-kDa N-recognin, encoded by UBR1, and the 20-kDa ubiquitin-conjugating enzyme encoded by UBC2. We report that both physical stability and functional activity of the N-recognin.Ubc2 complex require the presence of a highly acidic 23-residue region at the C terminus of Ubc2. Ubc2-C88A, an inactive variant of Ubc2 in which the active-site Cys-88 has been replaced by Ala, is shown to retain the affinity for N-recognin. Expression of Ubc2-C88A inhibits the N-end rule pathway, apparently as a result of competition between Ubc2 and Ubc2-C88A for binding to N-recognin. The two-hybrid (interaction cloning) technique was used to identify a approximately 170-residue C-terminal fragment of the 1,950-residue N-recognin as a Ubc2-interacting domain. We also show that the level of UBR1 mRNA decreases upon overexpression of UBC2. This effect of UBC2 is observed with cells whose UBR1 is expressed from an unrelated promoter but is not observed if UBR1 contains a frameshift mutation, or if the Ubc2 protein lacks its C-terminal acidic region. The N-recognin.Ubc2 complex appears to regulate the expression of N-recognin through changes in the metabolic stability of its mRNA.

Additional Information

© 1993 American Society for Biochemistry and Molecular Biology. User lisence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). (Received for publication, December 23, 1992, and in revised form, February 23, 1993). This work was supported by Grants AG08991 and GM31530 from the National Institutes of Health (to A. V.). 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. Supported by a postdoctoral fellowship from the American Cancer Society. Supported by a postdoctoral fellowship from the European Molecular Biology Organization. We thank the colleagues whose names are cited in the paper for their gifts of plasmids and strains. We are indebted to Bonnie Bartel for her advice as well as the gifts of strains and plasmids, and to Pei Pei Wu for her contributions to early immunoprecipitation experiments. We thank Christopher Byrd, Erica Johnson, Jennifer Johnston, and Irene Ota for comments on the manuscript.

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Published - J._Biol._Chem.-1993-Madura-12046-54.pdf


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