Fig 2: AF facilitates ubiquitin charging to E2s.The effects of AF on ubiquitin charging in vitro to UBA1 (a), UBE2G2 (b, c), and UBE2D1 (d). The assays were performed using purified recombinant proteins. 250 nM UBA1 alone or in combination with 4 µM His-E2s (His-UBE2G2 or His-UBE2D1) were treated with increasing concentrations of AF in reaction buffer for 1 min. ATP (50 µM) was added to initiate the reaction. The reactions proceeded at 15 oC for 45 s and stopped by adding non-reducing loading buffer. 5 mM DTT was used to disrupt the thioester bond that links ubiquitin to the active cysteine of E2s. Source data are provided as a Source Data file.

Fig 3: AF binds to the UFD of UBA1 and conjugates to cysteine 1039.a Effect of Iodoacetamide or DTT on the interaction of UBE2G2 with the UFD fragment of UBA1. b C1039A but not C1040A mutation diminishes AF-enhanced UBA1-UBE2G2 interaction in cells. HeLa cells transfected with the indicated plasmids were treated with 100 nM AF for 2 h, followed by processing for IP and IB. c C1039A but not C1040A mutation diminishes AF-enhanced MBP-UFD and UBE2G2 interaction in vitro. Recombinant MBP-UFD and its mutants were incubated with UBE2G2 along with different doses of AF. UBE2G2 bound to MBP-UFD, or its mutants was detected by IB. d Predicted binding complex of UBA1 with auranofin and E2G2. The binding interaction of the portion of Au-PEt3 of auranofin at the UFD domain is shown in a close-up panel. The protein structure of UBA1 (PDB 6DC6) and E2G2 (PDB 4LAD) are represented in ribbons. The gold Au (I) is coordinated to Cys1039 and rendered as balls in blue, the triethylphosphine ligand of auranofin in the pocket is shown in sticks. The domain structure of UBA1 is shown on top of the model. e Mutation of two predicted AF binding residues, E1037 and E1049, respectively, abrogates AF-enhanced UBA1-UBE2G2 interaction by coIP. Source data are provided as a Source Data file.

Fig 4: UBE2G2 catalytic activity is involved in proinsulin degradation.A. K562 cells stably expressing HLA-A*02:01 and PPI were transduced with a CRISPR-Cas9 vector containing a gRNA sequence directed against the N-terminal region of the UBE2g2 gene and selected with puromycin. Monoclonal knockouts were generated by limited dilution and analyzed for UBE2g2 expression. Genomic DNA was isolated and sequenced for the presence of insertions of deletions (indels) within the gRNA target region. Both UBE2g2 alleles were aligned to a reference sequence (NCBI gene entry 7327). B. K562 cells from (C) were retransduced with an empty cDNA vector (KO) or a gRNA-resistant cDNA vector encoding wt HA-UBE2G2 (WT), or a catalytically inactive mutant HA-UBE2G2 (C89S). Cells were sorted for mAmetrin to enrich for gRNA-positive cells. Next, cell lysates were analyzed by WB for expression of UBE2G2 and proinsulin. Human transferrin receptor (TfR) was used as a loading control. C. K562 cell lines from (B) were treated with 200ug/mL CHX for the indicated times, followed by WB analysis of total proinsulin levels. A representative blot from three independent experiments is shown. D. Results from 6 independent experiments of (C) were quantified, corrected for actin levels and normalized to t = 0 (Error bars = SD, paired Students’ T-test; *p<0,05; **p<0,005, ns = non-significant) Data points are shown in S2 Table. E. The HLA-eluted peptidome from (rescued) UBE2G2 K.O. clones from (B) and parental cells was analyzed by mass spectrometry (N = 1) and quantified from MASCOT output. Abundance of specific peptides and total (>35 ion scored) peptides is shown as a percentage of their presentation in UBE2G2 K.O. + WT UBE2G2 cells in a white-red gradient-coded table. See S3 Table.

Fig 5: An E2-specific CRISPR knockout screen identifies UBE2G2 to be involved in PI-GFP degradation.A. An arrayed CRISPR library targeting every known human E2-conjugating enzyme with three different gRNAs per gene [22] was transduced into K562 cells expressing PPI-GFP. Cells were selected with puromycin and GFP levels were assessed by flow cytometry 9 days post-infection. Results were quantified and compared to GFP levels in empty vector-expressing cells. For sample IDs, see S1 Table. B. Histograms of cells transduced with gRNAs targeting UBE2g2 or UBE2g1 (grey) or an empty vector control (red) from the screen shown in (A). PI-GFP levels were evaluated using flow cytometry. Raw values and calculations are shown in S2 Table.