Figure 1
Characterization of ubr11 mutants specifically defective in the recognition of N-terminal type 1 or type 2 residues. (a and b) Each strain was cultured with or without the indicated dipeptide for 4ย h. The relative fluorescence intensities of ArgNd-GFP and TrpNd-GFP, which are type 1 and type 2ย N-end rule substrates, respectively, were measured by flow cytometry. (a) The effect of Lys-Leu dipeptides was severely compromised in the ubr11-T2 mutant. ubr11-T2, which has type 2ย N-terminal residue-specific recognition defect (left and middle), or the ubr11โ (right) strains, which express ArgNd-GFP, were transformed with the indicated plasmid, and monitored for changes in the fluorescence intensity of ArgNd-GFP. (b) Ubr11-T1 was specifically defective in the recognition of type 1ย N-end residues. Each strain was examined as described in (a). (c) The type 1ย N-degron sequence in Rec8 did not promote Rec8c degradation in the ubr11-T1 strain. The X-Rec8c-GFP was expressed from the nmt promoter. The steady state levels of the type 1 substrate, Arg-Rec8c-GFP (lanes 1 and 2), and the type 2 substrate, Trp-Rec8c-GFP (lanes 3โ5), in the indicated strains were examined by immunoblotting with anti-GFP antibody. Cdc2: loading control. (d) Amino acid sequences of the N-degrons used in this study. The DYKDDDDK sequence shown in parenthesis is a FLAG tag epitope that was inserted before the GFP protein.