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. 2019 Apr 23;38(11):e100368. doi: 10.15252/embj.2018100368

Figure 5. Specific substrate–ligase interaction sites mediate SUMO‐SIM‐independent Euc1‐Slx5 binding.

Figure 5

  1. Euc181–183 binds to Slx5 in Y2H assays. Y2H assay performed as in Fig 4A. Serial dilutions were spotted for each plasmid combination, and cells were grown at 30°C for 2 days. The Slx5‐RING∆ construct is deleted for the Slx5 C‐terminus from the beginning of the RING domain (∆aa 488–619). See also Fig EV3B.
  2. Euc1 binds to Slx5 in vitro. Recombinant purified GST or GSTEuc1 was used in GST pull‐down assays to co‐precipitate recombinant 6HisSlx5.
  3. Euc1 binds to Slx5 in vivo. Cell lysates from an euc1∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids (under EUC1 promoter) were subjected to immunoprecipitation using anti‐FLAG beads. IP eluates were probed by WB with Slx5, SUMO, and Euc1 antibodies, and inputs were probed with Slx5, FLAG, and Dpm1 antibodies (top to bottom). Note that the Euc1‐Slx5 interaction is independent of the Slx8 ligase activity (slx8‐CD, lanes 5–8).
  4. Euc1 Slx5‐binding mutants (SBM1, SBM2) affect Euc1‐Slx5 interaction in Y2H assays. AD‐Euc181‐183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD‐Slx5 constructs as described in Fig 4A. Mutations: SBM1: aa 139–143 ENQKK>ANAAA, SBM2: aa 162–165 KEVF>AAAA. Serial dilutions were spotted, and cells were grown at 30°C for 2 days. See Appendix Fig S6A for expression levels.
  5. Euc1‐SBM constructs show defective Slx5 binding in vivo. Mutations described in (D) were introduced into full‐length 3FLAGEuc1 constructs (with or without the K231R mutation), and FLAG IPs were performed as described in (C). WB analysis showed strong Slx5‐binding defects for the SBM1/SBM2 and SBM1+2 constructs (top panel, immunoprecipitated Slx5). WBs were probed as in (C).
  6. The Slx5 middle domain (Slx5‐Md) is required for interaction with Euc1. Y2H assays with AD‐Euc181–183 and BD‐Slx5 constructs. Slx5‐Md: aa 201–335, Slx5‐Md∆: ∆aa 201–338. Serial dilutions were spotted, and cells were grown at 30°C for 4 days. See also Fig EV3C.
  7. Euc1‐SBM constructs show defective binding to the Slx5‐Md. Y2H assay with the indicated constructs as in (D). See Appendix Fig S6A for expression levels.
  8. Schematic representation of Euc1, Slx5, and Slx8, domain features, and interactions. Domains and protein lengths are drawn to scale, and numbers below each bar denote amino acid positions. The mapped interaction between Euc1 and Slx5 is indicated by an arrow. CC: coiled‐coil domain, SBM: Slx5‐binding mutant, S: SUMO, SIM: SUMO‐interacting motif.

Source data are available online for this figure.