Fig. 1.

RNF167 is an E3 ligase that ubiquitinates GluA2 and regulates its surface expression. (A) Schematic representation of the subset of RING-domain E3 ubiquitin ligases that localize to endosomes. Signal peptide, transmembrane domains, and RING domains are indicated. (B) Surface expression of GluA2 was evaluated by using FACS analysis of HEK293T cells coexpressing Flag-GluA2 and WT or RING domain mutant E3 ligases. Data are presented as the mean of GluA2 surface expression relative to WT (% ± SEM, n = 3 independent experiments). (C) WT, but not mutant, RNF167 ubiquitinates GluA2. HEK293T cells expressing Flag-GluA2 and RNF167 were lysed, and Flag-GluA2 was immunoprecipitated and immunoblotted for the presence of ubiquitin. The asterisk (*) represents a nonspecific band found in total cell lysates. (D) Immunoprecipitated HA-tagged WT RNF167 expressed in HEK293T cells was deglycosylated by using endoglycosydase H (endo H) or peptide-N-glycosydase F (PNGaseF) and immunoblotted by using RNF167 antibody. (E) Mobility profiles of RNF167 glycosylation mutants. Thirty micrograms of total proteins from HEK293T cells expressing RNF167 WT or mutants were analyzed by immunoblotting for RNF167. See also Fig. S1 A–C.