Figure 4.

Ulp2 Counteracts Siz1/2-Mediated SUMO Chain Formation, which Targets SUMOylated DDK for Cdc48 ATPase-Assisted Proteasomal Degradation

(A) SUMOylation of Cdc7 is mediated by the SUMO ligases Siz1 and Siz2. Shown is ^HisSUMO Ni PD from the cim3-1 mutant expressing ^3HACdc7 (WT) and cells additionally lacking the SUMO ligase Siz1, Siz2, or both or carrying the mms21-11 allele.

(B) The decreased levels of SUMOylated Dbf4 species in cim3-1 ulp2Δ are restored when, instead of ^HisSUMO, a lysine-less SUMO variant (KRall) is expressed.

(C) ^HisSUMO Ni PD from WT cells and a temperature-sensitive cdc48-6 mutant expressing ^3HADbf4 under the control of an endogenous promoter (pDBF4), grown to an OD₆₀₀ of 0.7 at 28°C and then shifted to 37°C for 3 h. SUMOylated Dbf4 species accumulate in the cdc48-6 mutant compared with WT cells.

(D) Dbf4 interacts in Y2H with Siz2, Slx5, and Ulp2 (catalytically dead Ulp2-C624S; Ulp2_CD) but not with its N-terminally truncated variant Ulp2_CD-N₄₀₀. Like Dbf4, Cdc48 interacts with Ulp2 depending on its N terminus. 8 mM 3-amino-triazole (3-AT) was added to reduce auto-activation of the HIS3 reporter gene.

(E) Interaction of Dbf4 with Siz2, Slx5, and Ulp2 is lost in the absence of Siz1 and Siz2. Dbf4 binding to Siz2 is Siz1-dependent.

See also Figure S4.