FIGURE 3.

Identification and quantification of DDI1 ubiquitination sites. (A) Relative LFQ intensity of all the proteins detected by MS demonstrate that DDI1-GFP is the most abundant protein (green) in the GFP pull-down samples, followed by ubiquitin (orange). 287 more proteins (blue) were identified with marginal intensities. (B) Comparison of the intensities recorded for the two most abundant proteins in UBE3A^WT and UBE3A^LD reveal that whereas the levels of DDI1 detected are similar in both conditions, more ubiquitin was detected in the presence of UBE3A^WT overexpression [t-test, ^∗∗p-value < 0.05, (mean ± S.E.M., n = 3)]. (C) Schematic illustration of DDI1-GFP sequence, its domains (blue, UBL, ubiquitin-like domain; orange, HDD, helical-double domain; red, RVP, retroviral protease domain; green, GFP, GFP-tag of DDI1 protein) and the localization of the ubiquitination sites detected by mass spectrometry. Additionally, the diGly-modified peptides identified, within the ubiquitinated lysine marked in red, and the intensity values measured in each condition (WT, UBE3A^WT and LD, UBE3A^LD) are also indicated. “∙” indicates that the ubiquitination site is not reported in PhosphoSitePlus database. DiGly peptides encompassing K133 and K161 were statistically more abundant upon UBE3A^WT overexpression [t-test, ^∗p-value < 0.05, (mean ± S.E.M., n = 3)]. (D) UBE3A-dependent ubiquitination of three distinct DDI1-GFP mutants (K77R, K133R, or K161R) was monitored by western blot after isolating by GFP-pulldown. In red it is illustrated the ubiquitination pattern of DDI1, and in green the unmodified version of DDI1-GFP. Quantification and statistical analysis of the ubiquitination was performed with Image-J after normalizing FLAG intensities to GFP levels. Mutation on DDI1 lysine 133 significantly [one-way ANOVA, ^∗p-value < 0.05, (mean ± S.E.M., n = 3)] abolishes its ubiquitination by UBE3A^WT.