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Published August 29, 2008 | Published
Journal Article Open

Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway


Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ~700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ~70 residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with Kd of ~1 microM, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides, but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of (cognate) dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically, and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1's type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

Additional Information

© 2008 the American Society for Biochemistry and Molecular Biology. Received for publication, April 3, 2008, and in revised form, May 19, 2008. Originally published In Press as doi:10.1074/jbc.M802583200 on June 19, 2008. We thank R. Deshaies and P. Bjorkman (Caltech, Pasadena, CA) for the use of Analyst Assay Detection System and BIAcore in their laboratories; S. W. Stevens and J. Abelson (Caltech) for the AK6967 antibody; and D. G. Myszka (University of Utah, Salt Lake City) for the CLAMP and Scrubber programs. We are grateful to former and current members of the Varshavsky laboratory, especially to C.-S. Hwang, for helpful discussions and comments on the manuscript. This work was supported, in whole or in part, by National Institutes of Health Grants DK39520 and GM31530 (to A. V.). This work was also supported by the Ellison Medical Foundation and the Sandler Program for Asthma Research. 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.

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