The ubr11 m6 mutant was tested first because the conserved aspartic acid inter acts with the type 1 N terminal amino acid of substrate proteins. Fur ther, a corresponding mutation in mouse Ubr1 specifically interferes with its ability to bind type 1 amino acids in vitro. However, its functionality selleck FTY720 in vivo was not tested. The mutant Ubr11 m6 protein was expressed in the ubr11 strain from a multicopy plasmid, and its functionality was monitored. Interestingly, the S. pombe ubr11 m6 mutant was able to degrade both type 1 and type 2 model substrates and induce peptide uptake. Another mutant, ubr11 T1, was then examined because a mutation of the corresponding residue in S. cerevisiae Ubr1 produced a mutant that was defective in targeting type 1 substrates.

This conserved glycine is also located near the residue critical for type 1 N end recognition. The type 1 model substrate, ArgNd GFP, was highly expressed in the S. pombe ubr11 T1 mutant, as in Inhibitors,Modulators,Libraries the control ubr11 strain. In contrast, the fluorescence intensity of the type 2 substrate, TrpNd GFP, was low and its level increased when the type 2 dipeptide, Tyr Leu, was added to strains expressing either wild type Ubr11 or Ubr11 T1. Therefore, the ubiquitin ligase activity of Ubr11 T1 was not impaired, and the ubr11 T1 mutant had a specific defect in type 1 residue recognition, but responded normally to the type 2 dipep tide. For reasons that are still unclear, the fluorescence intensity and protein levels of the type 2 substrate, TrpNd GFP, were partially increased when type 1 Lys Leu dipeptides were added to a strain expressing wild type Ubr11.

However, Lys Leu dipeptides were not effective in the Inhibitors,Modulators,Libraries ubr11 T1 mutant, confirming that this mutant had lost the ability to interact with type 1 N terminal amino acids. To exclude the possibility that the defect in the ubr11 T1 mutant was confined to the ArgNd N degron, the stabil ity of another N end rule substrate, bearing a different N degron, was examined. The C terminal fragment of Rec8, which is generated by a separase mediated cleavage of cohesin, is an endogenous substrate of the Arg N end rule pathway in Inhibitors,Modulators,Libraries S. pombe. Although the N degron sequence in Arg Rec8c is completely Inhibitors,Modulators,Libraries different from that in ArgNd, Arg Rec8c GFP, but not Trp Rec8c GFP, was stabilized in the ubr11 T1 mutant. Therefore, it was concluded that the ubr11 T1 mutant was defective in the recognition of N terminal type 1 amino acids in general.

Utilization of dipeptides by ubr11 mutants Inhibitors,Modulators,Libraries defective in recognizing enzyme inhibitor type 1 or type 2 residues Subsequently, the ability of the mutants to support pep tide uptake was tested. All proteins were expressed in the ubr11 host strain from a multicopy plasmid. Consistent with our previous report, ubr11 cells harboring the empty vector were defective in the up take of all dipeptides examined. Interestingly, the uptake of all three dipeptides was not affected in the ubr11 T1 mutant.