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, 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 approximately 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 approximately 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 approximately 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 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Zanxian
Z
Xia
Ailsa
A
Webster
Fangyong
F
Du
Konstantin
K
Piatkov
Michel
M
Ghislain
Alexander
A
Varshavsky
D020816:Amino Acid Motifs / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D004151:Dipeptides / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
C090457:CUP9 protein, S cerevisiae; D004151:Dipeptides; D018398:Homeodomain Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases