The N-end rule relates the in vivo half life of a protein to the identity of itsN-terminal residue. UBR1, the E3 of the N-end rule pathway in Sacchnromzycescerevisiae, targets proteins that bear destabilizing N-terminal residues for Ub-dependent,processive degradation. UBR1 binds protein substrates or dipetidesthrough two distinct sites: the type 1 site, specific for basic residues, and the type2 site, specific for bulky hydrophobic residues. UBR1 also recognizes an internaldegradation signal of the 35 kDa homeodomain protein CUP9, a transcriptionalrepressor of the di- and tripeptide transporter PTR2.
Here I report that the internal degradation signal of CUP9 is recognizedby UBR1 through its third, distinct substrate-binding site. Occupation of the type1 or type 2 sites of UBR1 by dipeptides allosterically stimulates theUBR1-dependent multi-ubiquitylation of CUP9 in an in vitro system, whichconsists of purified components of the yeast N-end rule pathway. UBR1 is thefirst E3 shown to be allosterically regulated by small compounds. Thisregulation underlies, in vivo, the accelerated UBR1-dependent degradation ofCUP9 in the presence of dipeptides with destabilizing N-terminal residues. Theresult is a positive feedback circuit that controls the peptide import inS. cerevisiae. Specifically, the imported dipeptides bind to UBR1 and acceleratethe UBR1-dependent degradation of CUP9, thereby derepressing thetranscription of PTR2 and increasing the cell's capacity to import peptides.
I also describe a new, autoinhibition-based molecular mechanismunderlying the activation of UBR1 by dipeptides. UBR1 is an autoinhibitedprotein, in that the binding of dipeptides to the type 1 and type 2 sites of UBR1enhances the dissociation of the C-terminal autoinhibitory domain of UBR1 from its substrate-binding N-terminal region. Moreover, this dissociation, whichallows the interaction between UBRl and CUP9, is strongly increased only ifboth type 1 and type 2 sites of UBRl are occupied by dipeptides. Anautoinhibitory mechanism discovered in the S. cerevisiae UBRl is likely to recurin metazoan homologs of UBRl, and may also be involved in controlling theactivity of other Db-dependent pathways.
