Fig 2: The AUP1–UBE2G2 interaction is required for ERAD.(A) Equimolar amounts of GST-AUP1 (aa 292 to 410) or GST were immobilized on glutathione beads and binding to the indicated in vitro translated 35S-labeled E2s was assessed. Left panel is 5% of input used for binding. E2s are referred to by their unique alpha-numeric designation (e.g., UBE2G1 and UBE2J1). (B) HT1080 or UBE2G2 KO cells were transfected with INSIG-1-MYC and degradation assessed by CHX chase. (C) The indicated cells were transfected with NHK-HA and empty vector, WT or catalytically inactive (C89S) N-terminally MYC-tagged UBE2G2, and NHK-HA degradation assessed. (D) Cells were transfected with NHK-MYC and HA-ubiquitin and ubiquitination of NHK assessed following proteasome inhibition and IP (right panel). Left panel is 10% of total cell lysate used for IP, indicating no discernable change in total ubiquitination. (E) Levels of endogenous HRD1 were monitored by CHX chase in the indicated cell types. (F) Ubiquitination of endogenous HRD1 was assessed following IP with HRD1 antiserum. Arrow in upper panel indicates migration of unmodified HRD1. Control immunoprecipitates using rabbit serum are shown for comparison. The data underlying this figure can be found in S1 Data. aa, amino acid; AUP1, ancient ubiquitous protein 1; CHX, cycloheximide; ERAD, endoplasmic reticulum–associated degradation; IP, immunoprecipitation; KO, knockout; WT, wild type.

Fig 3: The G2BR of AUP1 is required for ERAD of mutant ribophorin and GFPu.(A) HT1080 or HRD1 KO cells were transfected with plasmid encoding RI332-MYC and with plasmid encoding HRD1 as indicated. Cells were subject to CHX chase and RI332-MYC degradation monitored by western blot. GFP serves as a transfection efficiency control. (B) HT1080 or AUP1 KO cells were transfected with plasmid encoding RI332-MYC and AUP1-FLAG WT or deletion mutants assessed for RI332 degradation. Lower band (*) in MYC blot is nonspecific. (C, D) HEK293 or AUP1 KO cells were assessed for RI332 turnover as in (B). (E) HT1080 or AUP1 KO cells were transfected with GFPu plasmid, and its degradation assessed by emetine chase, which has previously been used to assess GFPu turnover [40]. Luciferase and β-actin serve as transfection efficiency and gel loading controls, respectively. (F) HT1080 AUP1 KO cells were transfected with GFPu and the indicated AUP1-FLAG WT or deletion mutants, and GFPu degradation assessed as in (E). The data underlying this figure can be found in S1 Data. AUP1, ancient ubiquitous protein 1; CHX, cycloheximide; ERAD, endoplasmic reticulum–associated degradation; G2BR, UBE2G2 Binding Region; KO, knockout; WT, wild type.

Fig 4: Disruption of UBE2G2:G2BRAUP1 interaction abrogates AUP1 function in cells.(A) GST or indicated GST-AUP1 fusions (aa 292 to 410, expression shown in Coomassie-stained gel) were immobilized on glutathione beads and binding to 35S-UBE2G2 was assessed as in Fig 3A. Mutations include 2A (R382A/Q383A), 2E (R382E/Q383E), and 5E (R382E/Q383E/K390E/R398E/R400E). (B) AUP1 KO cells were transfected with NHK-HA and forms of full-length AUP1-FLAG with the indicated point mutations from (A) and NHK degradation assessed by CHX chase. (C) Binding assay performed as in (A) with AUP1 mutants predicted to disrupt hydrophobic interactions with UBE2G2. (D) Cellular assay performed as in (B) with AUP1-FLAG mutants that disrupt interactions with UBE2G2 in vitro in (C). The data underlying this figure can be found in S1 Data. aa, amino acid; AUP1, ancient ubiquitous protein 1; CHX, cycloheximide; G2BR, UBE2G2 Binding Region; KO, knockout; WT, wild type.

Fig 5: G2BRAUP1 protects UBE2G2 from degradation.(A) Indicated cells were assessed for steady state levels and turnover of endogenous UBE2G2 by CHX chase. (B) Transcript levels of endogenous UBE2G2 in AUP1 KO cells compared to HT1080 as determined by qPCR. Mean and standard deviation from three independent experiments is shown. (C) Turnover of transfected MYC-UBE2G2 or a catalytically inactive mutant (C89S) was monitored in AUP1 KO cells. (D) AUP1 KO cells were transfected with plasmids encoding MYC-UBE2G2 and GFP fusions of either a scrambled G2BR (SCR), G2BRgp78, or G2BRAUP1, and the turnover of UBE2G2 was assessed. (E) Relative abundance of gp78 (left panel) and AUP1 (right panel) in HT1080 cells was determined by resolving indicated amounts (in pmoles) of purified GST-gp78 (aa 429 to 611) or GST-AUP1 (aa 61 to 410) fusions alongside approximately 1 × 104 cellular equivalents of cell lysate and immunoblotting with gp78 or AUP1 antibodies. For each antibody, the amount of immunoreactivity in the lysate was compared to a defined quantity of a GST fusion protein of gp78 or AUP1 spanning the epitope recognized by the probing antibody. This analysis suggests there is close to 40-fold as much cellular AUP1 (150 pmole) as gp78 (4 pmol). The data underlying this figure can be found in S1 and S8 Data. aa, amino acid; AUP1, ancient ubiquitous protein 1; CHX, cycloheximide; G2BR, UBE2G2 Binding Region; KO, knockout; qPCR, quantitative polymerase chain reaction; WT, wild type.