yeast two-hybrid assay ultimate y2h screen  (Hybrigenics sa)

Journal: Molecular Cell

Article Title: SUMO-Chain-Regulated Proteasomal Degradation Timing Exemplified in DNA Replication Initiation

doi: 10.1016/j.molcel.2019.08.003

Figure Lengend Snippet: 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 His SUMO Ni PD from the cim3-1 mutant expressing 3HA Cdc7 (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 ulp2 Δ are restored when, instead of His SUMO, a lysine-less SUMO variant ( KRall ) is expressed. (C) His SUMO Ni PD from WT cells and a temperature-sensitive cdc48-6 mutant expressing 3HA Dbf4 under the control of an endogenous promoter ( pDBF4 ), grown to an OD 600 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 -N 400 . 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 .

Article Snippet: ULTImate yeast two-hybrid ( Y2H) screen , Hybrigenics Services , https://www.hybrigenics-services.com/.

Techniques: Mutagenesis, Expressing, Variant Assay, Control, Activation Assay, Binding Assay

Journal: Molecular Cell

Article Title: SUMO-Chain-Regulated Proteasomal Degradation Timing Exemplified in DNA Replication Initiation

doi: 10.1016/j.molcel.2019.08.003

Figure Lengend Snippet: SUMO-Interacting Motifs at the N terminus of Ulp2 Mediate Binding to Dbf4 and Are Required to Protect SUMOylated DDK from Proteasomal Turnover (A) Predicted SIMs in the Ulp2 N terminus resemble confirmed Slx5 SIMs in number and relative positioning (highlighted green). Shown is a schematic representation of Slx5 and Ulp2 with the RING and protease domains, SIMs (blue), and introduced mutations (red) that disrupt potential SIMs. (B) Y2H interaction of Dbf4 with Ulp2 is abolished when either first three or all five N-terminal putative SIMs of Ulp2 are mutated, similar to Ulp2 CD -N 400 . (C and D) Recombinant GST fusion of the Ulp2 N terminus (aa 1–400) binds both yeast-free SUMO (C) and human poly-SUMO3 chains (D) in vitro , with a stronger preference for chains having more than 5 SUMO3 moieties. (E) ulp2-sim mutants phenotypically resemble ulp2 Δ cells regarding slow growth and temperature sensitivity but have lower sensitivity to HU. (F) Both ulp2-sim mutants similar to ulp2 Δ fail to protect SUMOylated Dbf4 against SUMO chain/STUbL-mediated proteasomal degradation. Shown is His SUMO Ni PD from cim3-1 , cim3-1 ulp2 Δ, and cim3-1 cells carrying either Ulp2 9PK or its SIM mutant variants. See also Figure S5 .

Article Snippet: ULTImate yeast two-hybrid ( Y2H) screen , Hybrigenics Services , https://www.hybrigenics-services.com/.

Techniques: Binding Assay, Recombinant, In Vitro, Mutagenesis