Figure 6. E2^HTH substitutions differentially impact substrate ubiquitination by the E3 APC/C.

• A, B
  Schematic of UBE2S~Ub (A) and UBE2C~Ub interactions (B) with the APC/C. UBE2S~Ub uses its C‐terminal extension to bind to a groove formed by APC2 and APC4. The UBE2S^HTH interacts with APC2 and the APC11 RING domain positions the substrate‐linked Ub for chain elongation. UBE2C~Ub is positioned by the APC11^RING and APC2 winged‐helix B (WHB) domains during substrate ubiquitination (Brown et al, 2015, 2016).
• C
  Securin ubiquitination by APC/C and either UBE2C WT or K157E, resolved by SDS–PAGE and fluorescent scanning. Quantification of 4 or more ubiquitins added on the substrate (right) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch’s t‐test (****P ≤ 0.0001, n = 5 independent, technical replicates). Error bars: SEM.
• D
  Similar to C, Ub‐securin ubiquitination by APC/C and either UBE2D2 WT or charge‐swap variants. Quantification of 4 or more ubiquitins (right) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one‐way ANOVA (****P ≤ 0.0001, n = 4 independent, technical replicates). Error bars: SEM.
• E
  Representative fluorescent scan of an SDS–PAGE gel of APC/C‐dependent polyubiquitination of Ub‐CyclinB* by a panel of UBE2S^HTH charge‐swap variants. Quantification in Fig EV5C.
• F
  The recombinant UBE2S ER/ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild type (WT), monitored by immunoblot. G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA (siFF) or two UBE2S siRNAs (siUBE2S).

Source data are available online for this figure.