Fig. 3: UBE3C induces VPS34 K29/K48-branched ubiquitination, leading to an enhanced VPS34 proteasomal degradation.
a Reciprocal immunoprecipitation analysis of the interaction between UBE3C and VPS34 in 293T cells. b In vitro interaction of baculovirally purified Flag-VPS34 with separately purified HA-UBE3C. c Analysis of VPS34 ubiquitination in 293T cells transfected with indicated constructs. VPS34 was immunoprecipitated by M2 beads, followed by western blot analysis with indicated antibodies. The knockdown efficiencies of UBE3C shRNAs are shown in j. d–f Analysis of VPS34 ubiquitination in 293T cells transfected with indicated constructs. g In vitro ubiquitination of Flag-VPS34 purified from 293T cells in the presence or absence of baculovirally purified UBE3C. The reaction mixture was analyzed by Western blot with indicated antibodies. h Quantification of indicated ubiquitin species identified by intact MS analysis on in vitro ubiquitinated and Lbpro*-treated VPS34. Data are means from two independent experiments. The representative spectrum of intact MS data is shown in Supplementary Fig. 3d. i Ubiquitin linkage types and their abundance identified by LC–MS/MS on VPS34 ubiquitinated in vitro in the presence or absence of UBE3C and treated with Lbpro*. Peak intensities from two independent experiments are shown. j, k Western blot analysis of VPS34 levels in 293T cells stably expressing UBE3C shRNAs (j) and treated with MG132 (k). l Schematic representation for the ubiquitin chain type switch in K29R ubiquitin replacement cells after doxycycline treatment. m Immunoprecipitation analysis for the binding of proteasome subunit S5A with ubiquitinated VPS34 in wild type or K29R ubiquitin replacement cells transfected with indicated constructs and treated with or without doxycycline. n, o Western blot analysis of VPS34 levels in K29R ubiquitin replacement cells transfected with or without UBE3C siRNA and treated with doxycycline for at least 96 h together with cycloheximide for indicated time points (n) or MG132 for 16 h (o). The knockdown efficiency of UBE3C siRNA is shown on the right panel of n. Source data are provided as a Source Data file.
