N-end Rule Specificity within the Ubiquitin/Proteasome Pathway Is Not an Affinity Effect

Abstract

The N-end rule relates the amino terminus to the rate of degradation through the ubiquitin/26 S proteasome pathway. Proteins bearing basic (type 1) or large hydrophobic (type 2) amino termini are assumed to be targeted through this pathway by their higher affinity for binding to the responsible E3 ligase compared with proteins bearing other residues (type 3). Paradoxically, a significant fraction of eukaryotic protein degradation occurs through the N-end rule pathway, although the majority of cellular proteins are type 3 substrates. We have exploited specific interactions between ubiquitin carrier proteins (E2/Ubc) and their cognate E3 ligases to purify for the first time the mammalian N-end rule ligase E3␣/Ubr1 to near homogeneity. In vitro studies show that E3␣ forms lysine 48-linked polyubiquitin degradation signals on type 1-3 substrates and is absolutely dependent on Ubc2/Rad6 orthologs. Biochemically defined kinetic studies show that the basis of N-end rule specificity is a k cat rather than the K m effect originally proposed, since all three substrate classes show similar binding affinities (K m ϳ5 M) but V max values that are 100-and 50-fold greater for type 1 and 2 versus type 3 model substrates, respectively. In addition, the N-end rule dipeptides lysylalanine and phenylalanylalanine are general noncompetitive inhibitors for E3␣-catalyzed ubiquitination of type 1-3 substrates rather than typespecific competitive inhibitors as predicted. These observations are consistent with a model in which the N-end rule effect reflects substrate binding-induced transitions in E3␣ to a catalytically competent conformer, the equilibrium for which depends on the identity of the amino terminus or the presence of basic or hydrophobic surface features. The model reconciles conflicts between specific predictions and empirical observations relating N-end rule targeting in addition to explicating the efficacy of selected dipeptides as potent in vivo inhibitors of this pathway.

Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain

“…This conclusion, based on the authors' interpretation of their enzymological evidence (29), is directly in conflict with both the earlier UBR1-binding data (6) and the results above. The technically straightforward, catalysis-free binding assays with yeast and mouse UBR1 (Fig.…”

“…Recently, Baboshina et al (29) suggested that the mammalian N-end rule pathway distinguishes between stabilizing and destabilizing N-terminal residues not through differences in the thermodynamic affinities of UBR1 (E3␣) for these residues, but largely through differences in the catalytic activity of UBR1 in response to its (comparably avid) binding to either stabilizing or destabilizing N-terminal residues. This conclusion, based on the authors' interpretation of their enzymological evidence (29), is directly in conflict with both the earlier UBR1-binding data (6) and the results above.…”

Tripartite Motif Ligases Catalyze Polyubiquitin Chain Formation through a Cooperative Allosteric Mechanism

“…4A). The latter control additionally confirms that the assays contain sufficient Uba1 to maintain all Ubc5A charged as the corresponding Ubc5A-125 I-ubiquitin thioester, the actual substrate for TRIM32 (33,49). Excision of the lanes from the gel and quantitation of the associated radioactivity by ␥-counting allows one to calculate the corresponding rate of polyubiquitin chain formation based on the specific radioactivity of the 125 Iubiquitin (10,32,49).…”

The E2 Ubiquitin-conjugating Enzymes Direct Polyubiquitination to Preferred Lysines

“…In line with these reports, we show that in vitro E2D can utilize both Lys 48 and Lys 63 in polyubiquitination. A broader range of lysine specificity was detected when the Rad6 (E2A/B) was used in polyubiquitination with various E3s (Bre1, Ubr1, and Rad18) (20,34,45), each selecting different lysine specificity from the potential conjugation abilities of Rad6 (Lys 11 and Lys 48 ). In aggregate, the clear overlap in the lysine preferences demonstrated by a given E2, independent of the presence or absence of an E3 enzyme, is a strong indicator of the central role of the E2 enzymes in the determination of the lysine preference in ubiquitination.…”