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

Figure 6. Specific Euc1‐Slx5 interactions are required for ub‐hotspots and Euc1 ubiquitylation.

Figure 6

  1. Euc1 Slx5‐binding mutations impair Euc1 ubiquitylation. His‐ubiquitin‐modified proteins were enriched by denaturing NiNTA‐PDs as described in Fig 4E. 3FLAG‐tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter. NiNTA‐PD eluates were probed with FLAG and ubiquitin (P4D1) antibodies. Whole cell lysates (input) were probed with FLAG and Dpm1 antibodies. Asterisk denotes a non‐specific band.
  2. The Slx5‐SIMs and Slx5‐Md are required for Euc1 ubiquitylation. NiNTA‐PDs for His‐ubiquitin as in (A). All strains expressed wild‐type 3FLAGEuc1 from plasmids under the ADH promoter and His‐ubiquitin. Slx5 constructs were expressed from plasmids under the control of the endogenous promoter. WBs were probed as described in (A), and Slx5‐levels were probed using an HA antibody. Slx5‐SIM*: SIMs 1–4 were mutated as described (Xie et al, 2010); for SIM5, aa 477–479 (IIV) were mutated to alanines. To avoid possible mislocalization by deletion of the Slx5‐Md, which overlaps with a putative NLS (Westerbeck et al, 2014), an N‐terminal NLS was fused to all constructs. See Appendix Fig S7C for HU complementation.
  3. Euc1 Slx5‐binding mutations (SBM1/SBM2) reduce/abolish ub‐HSs and recruitment of Slx5 to ub‐HSs. ChIP‐qPCR for ub‐K48 (top) or Slx5 (using Slx5 antibody, bottom) in euc1∆ ubx5∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter. Data represent means ± SD (n = 3). See also Appendix Fig S7E–G.
  4. Slx5‐SIMs and the Slx5‐Md are required for the formation of ub‐HSs and recruitment of Slx5. ChIP‐qPCR for ub‐K48 (left) or Slx5 (anti‐HA ChIP, right) in ubx5∆ cells or slx5∆ ubx5∆ cells complemented with plasmids expressing the indicated Slx5 constructs under the control of the SLX5 promoter. Data represent means ± SD (n = 2). See also Appendix Fig S7H.
  5. Schematic model for the proposed sequence of events at ub‐hotspots. See main text (Discussion) for details. SIM: SUMO‐interacting motif, Md: middle domain, S: SUMO, Ub: ubiquitin.

Source data are available online for this figure.