Fig. 6: The effects of key residue mutations of FL PRT1 and the third residue of N-degron.
a Isothermal titration calorimetry (ITC) results of PRT1 mutants (PRT1Y317A and PRT1I333A) with the YKA peptide. No affinity or very weak affinity were determined. b ITC results of PRT1 mutants (PRT1Y317A, PRT1I333A, PRT1Y317A/I333A, and PRT1F352A) with the YKF peptide. KD values: PRT1Y317A = 41.3 ± 0.799 μM; PRT1I333A = 13.7 ± 1.68 μM; PRT1Y317A/I333A = N.D. (not determined); PRT1F352A = N.D. The detailed parameters are listed in Supplementary Table 3. c Ubiquitylation activity of FL PRT1 depending on reaction time (each lane at 0, 0.5, 1, 3, 5, and 7 h). The poly-Ub pattern of Tyr61-BBV198E was detected in the presence (left) and absence (right) of PRT1. See Supplementary Fig. 9 for the details of the diminished auto-ubiquitylation activity of the Tyr61-BBV198E. d Ubiquitylation activity of the PRT1 mutants with an authentic Nt sequence-containing substrate, YKF-BBV198E. Ctrl: no PRT1 protein. e Ubiquitylation activity of the PRT1 mutants with an Nt permutated substrate, YAA-BBV198E (alanine at the second and third position). The key residue mutants of PRT1 showed reduced or no ubiquitylation activity compared with that of the WT. These loss-of-function effects were more reinforced when the N-terminus of Tyr61-BBV198E was permutated to YAA. Ctrl: no PRT1 protein. The ubiquitylation reactions are quenched at 3 h. All images probed with anti-His Ab were detected under intensive conditions (2.5× loading volume and longer exposure time) compared to those probed with anti-FLAG or anti-Ub. The original western (same loading volume and exposure intensity) images with anti-His Ab are shown in Supplementary Fig. 10b. These ubiquitylation assays were performed at least three times.
