Topological characterization of the E2–E3 network. (A) Venn diagram showing the shared E2–E3 interactions in the 75th percentile selected LEU2-set and the 50th percentile selected LacZ-set and the overlap between them. (B) Distribution of the E3 connectivity for each individual E2. Depicted are the numbers of E3 interactions observed in the shared LacZ- and LEU2-set among all interactions of a single E2. (C) Distribution of the E2 connectivity for each individual E3, idem as panel B.

Mutant UbcH5B and mutant CNOT4–N63 interactions. (A) Overview of RING E3 interaction patterns between wild type and UBE2D2(UbcH5B) (K63E). E3s in purple interacted with both wild type and mutant UBE2D2(UbcH5B), in green only with wild-type UBE2D2(UbcH5B) and in pink only with the K63E mutant. (B) E2 interaction pattern of wild type and mutant CNOT-N63. White indicates no interaction, purple or green an interaction with CNOT4-N63 or CNOT4-N63 D49K/E49K, respectively.

UBE2U physically interacts with MDM2. (A) HEK293T cells were transiently transfected with myc-tagged MDM2, lysed and incubated with GST–E2s immobilized on gluthathion-agarose beads. Bound material was resolved on 7.5% SDS–PAGE and proteins were visualized after immunoblotting with antibodies against myc. (B) Untransfected U2OS cells were lysed and combined with GST–E2s as in panel A. Immunoblotting was carried out using MDM2 antibodies (upper panel) and using p53 antibodies after reprobing (lower panel). Arrow indicates MDM2 signal, asterisk indicates background band.

Design of a UBE2N derivative with a new E3 interaction specificity. (A) Sequence alignment of UBE2D2(UbcH5B) and UBE2N(Ubc13). Secondary structure elements (indicated on top in purple and green for the UBE2D2-specific extension of helix 1) and regions involved in E3 RING interactions were derived from the E2 crystal structures (PDB codes 2ESK and 1JBB for UBE2D2 and UBE2N, respectively). Amino acids are coloured according to their physico-chemical properties. (B) E3 interaction patterns of UBE2D2, UBE2N and UBE2N mutants. The library of 251 yeast clones expressing B42–RING fusions were re-arrayed on three 96-well plates and mated with LexA–E2 expressing yeast clones. Diploids were selected and interactions were visualized as described in Figure 1. Pictures were taken after 72 h incubation at 30°C. Upper and lower three squares in the outer two columns of each plate represent plate controls. (C) Quantification of wild type and mutant E2–E3 interactions. Spot staining (LacZ) and spot sizes (LEU2) of each E2–E3 interaction were quantified and corrected for background signals. Individual E2–E3 interactions are depicted as bars; black bars indicate no interaction and coloured bars indicate E2–E3 interactions. (D) Venn diagram showing the overlap in E3 interactions between the different E2 enzymes. (E) Expression of wild type and mutant E2 enzymes. Total cell lysates of yeast cells expressing the indicated LexA–E2 fusions were separated on 12.5% SDS–PAGE and analysed by immunoblotting using antibodies against LexA or tubulin (TUB1) as loading control.

Outline array based, mating yeast two-hybrid matrix screen. (A) Organization of LexA–E2 fusions as three independent bacterial triplicate clones and an empty LexA vector. (B) LexA–E2 protein expression levels. Total protein lysates of yeast cells transformed with LexA–E2 fusions were resolved on SDS–PAGE, transferred to membranes and probed with either LexA antibody or yeast tubulin (TUB1). (C) Overview of experimental yeast two-hybrid screening procedure. EGY48α cells were transformed with 36 LexA–E2 fusions and EGY48a cells were transformed with 250 B42–RING-fusions. Screening for pair-wise E2–E3 interactions was carried out by standardized array-based mating between EGY48α and EGY48a cells on YPD for 24 h at 30°C, followed by diploid selection on SC HWU⁻ for 48 h at 30°C. Interactions were visualized by transferring diploids on SC HWU⁻ X-Gal (colorimetric selection) or on SC HWUL⁻ (auxotrophic selection) under both B42-inducing (galactose as main carbon source) and repressive (glucose) conditions. Digital images of the interactions were taken at 3, 24-h interval time points. (D) Overview of standardized LexA–E2 array. Each spot represents EGY48α cells transformed with the indicated LexA–E2 fusion construct. Spots with a red square contained LexA–UBE2D2 and LexA–UBE2D2 K63E constructs and were mated throughout the entire procedure with EGY48a cells transformed with B42–CNOT4 N63 as positive and negative interaction controls, respectively. Spots with a green square were transformed with LexA–Caf40 and LexA–CNOT2-15 and served as auto-activating controls. Blue squared spots represent either EGY48a cells transformed with LexA-empty vector and an untransformed EGY48α strain, as mating/growing controls.

E2–E3 network. A network graph of binary E2–E3 domain interactions involving 124 E2 and E3 domains linked through 346 interactions. Nodes (domains) are shown as circles and interactions between them as black lines. E2 domains are shown as red circles and E3 domains as blue circles. The size of each node is linear related to the number of links of that node. Red square depicts E2 hub proteins, blue square hub E3s. Figure was generated using Cytoscape v.2.6.1.

Quality of the E2–E3 network. Physical E2–E3 interactions in relation to literature-curated, functional E2–E3 pairs. Hub node E3s were selected and interactions with E2s were scored when biochemically tested in in vitro ubiquitination assays as reported in literature.

GST-pull-down analysis between (hub) E2 and E3 enzymes. To validate yeast two-hybrid interactions, GST-pull-down analysis was performed between ten E2 enzymes and six E3 ligases. Approximately 10 μg GST–E2 enzyme were immobilized on G/A-beads and incubated with crude lysates expressing 6xhis–RING constructs. Bound proteins were resolved on SDS–PAGE, immono blotted and proteins were visualized using anti-6xhis antibodies. GST–E2 levels were visualized using Coomassie brilliant blue staining. An asterisk indicates a specific signal.