Figure 3: Functional analysis of the vWA:Ub-binding patch.
From: Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism
(a) Scheme showing a bacterial genetic selection system for ubiquitylation. (b) Ubiquitylation-addicted bacterial growth on selective (+Trimethoprim) or non-selective plates. A single scan of the plates 98 h post seeding is shown (for time-lapse live-scan movies, see Supplementary Movies 1 and 2). (c) Quantification of ubiquitylation-dependent bacterial growth. Average density of individual spots monitored by scanning the plates in 1-h intervals was plotted. Efficiency was calculated as the maximum growth density divided by the time of half max growth. NSG, no significant growth. (d) A representative SPR response curves for the vWA:Ub complex. (e) Single model binding analysis of SPR affinity measurements of Rpn10:Ub wt and the indicated mutants. Kd values are indicated (right; NB, no binding). (f) Comparison between the relative growth of wt or mutant spots and the SPR measured association constants. Pearson product-moment correlation coefficient is equal to 0.955. (g) A representative SDS–PAGE comparing the ubiquitylation amounts in purified wild-type and GST-Rpn10 mutant at the non-covalent Ub-binding patch. (h) Mass spectrometry analysis of the ubiquitylation sites for full-length wild-type Rpn10 and a mutant at the vWA:Ub non-covalent-binding patch. The peaks were scaled according to the relative quantities of the detected GG peptides. For each ubiquitylation site, the total number of detected GG peptides and their percentage in the population are shown (Supplementary Figs 3 and 4).
