HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 35, 24011-24028, August 29, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/35/24011    most recent M802583200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xia, Z. Articles by Varshavsky, A. Search for Related Content PubMed PubMed Citation Articles by Xia, Z. Articles by Varshavsky, A. Social Bookmarking                      What's this?

Substrate-binding Sites of UBR1, the Ubiquitin Ligase of the N-end Rule Pathway^*

Zanxian Xia¹, Ailsa Webster, Fangyong Du^¶, Konstantin Piatkov, Michel Ghislain^||, and Alexander Varshavsky²

From the Division of Biology, California Institute of Technology, Pasadena, California 91125, Celltech R&D, 216 Bath Road, Slough SL1 4EN, United Kingdom, the ^¶Department of Microbial Pathogenesis, Yale University, New Haven, Connecticut 06536-0812, and ^||Group of Physiological Biochemistry, University of Louvain, Croix du Sud 5/15, B-1348 Louvain-la-Neuve, Belgium

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, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and 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 a K_d of 1 µM, 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 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

Received for publication, April 3, 2008 , and in revised form, May 19, 2008.

^* 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.

¹ Present address: Center for Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, CA 90033.

² To whom correspondence should be addressed. Tel.: 626-395-3785; Fax: 626-440-9821; E-mail: avarsh{at}caltech.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C.-S. Hwang, A. Shemorry, and A. Varshavsky Two proteolytic pathways regulate DNA repair by cotargeting the Mgt1 alkylguanine transferase PNAS, February 17, 2009; 106(7): 2142 - 2147. [Abstract] [Full Text] [PDF]

				T. Tasaki, A. Zakrzewska, D. D. Dudgeon, Y. Jiang, J. S. Lazo, and Y. T. Kwon The Substrate Recognition Domains of the N-end Rule Pathway J. Biol. Chem., January 16, 2009; 284(3): 1884 - 1895. [Abstract] [Full Text] [PDF]

				A. Varshavsky Discovery of Cellular Regulation by Protein Degradation J. Biol. Chem., December 12, 2008; 283(50): 34469 - 34489. [Full Text] [PDF]

				C.-S. Hwang and A. Varshavsky Regulation of peptide import through phosphorylation of Ubr1, the ubiquitin ligase of the N-end rule pathway PNAS, December 9, 2008; 105(49): 19188 - 19193. [Abstract] [Full Text] [PDF]

				Z. Xia, G. C. Turner, C.-S. Hwang, C. Byrd, and A. Varshavsky Amino Acids Induce Peptide Uptake via Accelerated Degradation of CUP9, the Transcriptional Repressor of the PTR2 Peptide Transporter J. Biol. Chem., October 24, 2008; 283(43): 28958 - 28968. [Abstract] [Full Text] [PDF]