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

Figure EV1. Related to Figs 1 and 2. Seven ubiquitin hotspots across the yeast genome share a Cdc48‐dependent extraction mechanism and recruit Ymr111c/Euc1.

Figure EV1

  • A
    The FK2 ubiquitin antibody and a ub‐K48 specific antibody detect the same ub‐HSs in genome‐wide ChIP‐chip experiments. 16‐kb windows from genome‐wide ChIP‐chip data using either the ubiquitin (FK2) or ub‐K48 (clone Apu2) antibodies are shown. For comparison, data for ubiquitin (FK2) ChIP in WT, cdc48‐6, cdc48‐3 are reproduced from Fig 1B and for h2b‐K123R partially (ub‐HS4) from Fig 1A. Data represent means from two independent replicates.
  • B
    Table summarizing the ub‐HSs identified in Fig 1B. Stretches were defined by significantly enriched regions in ub‐K48 ChIP‐chip in cdc48‐6 (all except ub‐HS2) or Slx8‐9myc‐enriched regions in the cdc48‐3 background (ub‐HS2).
  • C, D
    Ubiquitin signals increase around 5–10‐fold in both cdc48‐6 and cdc48‐3 mutants with ubiquitin (FK2) and ub‐K48‐chain‐specific antibodies. ChIP‐qPCR experiment using ubiquitin (FK2) (C) and ub‐K48 (D) antibodies and indicated strains. Data represent means ± SD (n = 3 for (C), n = 4 for (D)).
  • E
    ufd1‐2, ubx4∆, and ubx5∆ show an increase of ubiquitin conjugates at all ub‐HSs similar to cdc48‐6. Genome‐wide ChIP‐chip data using the ub‐K48 or a non‐specific IgG control antibody for the indicated strains. Data for WT and cdc48‐6 reproduced from (A) for comparison. Data represent means from two independent replicates.
  • F
    A single point mutation within the ub‐HS‐motif abolishes ubiquitin enrichment. A G>T mutation was introduced in one of the conserved TTGTT repeats of ub‐HS4 F7 (bottom scheme) and integrated at the LEU2 locus as described in Fig 2A. ChIP‐qPCR for ub‐K48 demonstrated that the ubiquitin enrichment is lost upon mutation of the ub‐HS‐motif (ub‐HS4 F7‐mut). Experiments were performed in cdc48‐6 strains. Data represent means ± SD (n = 5).
  • G
    Table summarizing the confirmed hits from two independent Y1H screens as described in Fig 2D. Protein start sites are indicated. aa: amino acid, N.D.: not determined.
  • H
    Endogenous Euc1 does not bind the mutated ub‐HS4‐motif. ChIP with an Euc1‐specific antibody was performed in strains with ub‐HS4 F7 or ub‐HS4 F7‐mut integrated at the LEU2‐locus as described in (F). Experiments were performed in cdc48‐6 strains. Data represent means ± SD (n = 2).