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

Figure 4. Euc1 is SUMOylated, recruits Slx5 to ub‐hotspots, and is ubiquitylated in a Slx5/Slx8‐dependent manner.

Figure 4

  • A
    Euc1 interacts with SUMO pathway proteins in a yeast two‐hybrid (Y2H) assay. A Y2H reporter strain (PJ69‐7a) was transformed with Gal4 DNA‐binding domain (BD) and Gal4 activation domain (AD) fusion constructs in the indicated combinations. AD‐SUMO‐GG can be conjugated to SUMOylation substrates, while AD‐SUMO‐AA is conjugation‐deficient. Cells were spotted on control media or selective media (‐ His) and grown for 3 and 6 days, respectively.
  • B
    Euc1 is SUMOylated by Ubc9, Siz1, and Siz2 on lysine 231. Euc1 was immunoprecipitated from the indicated strains, and eluates were probed by WB against SUMO (top) and Euc1 (bottom). An up‐shifted band corresponding to Euc1SUMO was detected with both antibodies. Asterisks denote non‐specific bands.
  • C
    Euc1 is predominantly monoSUMOylated on lysine 231. Denaturing NiNTA pull‐downs (NiNTA‐PD) with strains expressing His‐tagged SUMO (HisSUMO) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter. Covalently SUMO‐modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1. Eluates were probed for SUMO to control for equal pull‐down, and Pgk1 was probed as input control. Euc1‐KR denotes the K231R mutation here and hereafter.
  • D
    Euc1 SUMOylation is required for ub‐HS formation. Ub‐K48 ChIP quantified by qPCR for the indicated strains. The ubx5∆ background was used to enhance the ubiquitin signal, and similar results were obtained in a WT background. Data represent means ± SD (n = 3).
  • E
    Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation. Denaturing NiNTA‐PDs as described for (C) but with strains expressing His‐ubiquitin (HisUbi) and 3FLAGEuc1 under the control of the ADH promoter. WBs were developed with a FLAG antibody to probe for 3FLAGEuc1, a ubiquitin blot served as PD‐control and Dpm1 as input control. The slx8‐CD allele carries the C206S and C209S mutations (Xie et al, 2007).
  • F, G
    SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub‐HSs. ChIP against Slx5 and Slx8 in the indicated genetic backgrounds was quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA‐Slx5 slx8∆), but not vice versa (Slx8‐9myc slx5∆). Data for WT and 3HA‐Slx5 in (F) and WT and Slx8‐9myc in (G) are from Fig 1D and are shown here for comparison. Data represent means ± SD (n = 2).

Source data are available online for this figure.