Fig. 3: Cullin8 complex promotes Mms4 ubiquitylation and regulates the Mms4 phosphorylation cycle and turnover.

a Time course experiment analyzing Mms4 species within one cell cycle in WT and cul8Δ cells. Logarithmically (log) grown Tc-HA-Mms4 cells of the indicated genotype were synchronized in G1 phase with α-factor (αF) and then released in YPD medium containing 1 mM Tetracycline (Tet). After cells reached G2/M, α-factor was added to the culture to arrest cells in the next G1 phase. Samples were taken at the indicated timepoints and the presence of HA-tagged unphosphorylated (^HAMms4) or phosphorylated Mms4 (^HAMms4-P) species was analyzed by western blot. Immunodetection of Pgk1 served as a loading control. The percentage of phosphorylated Mms4 compared to total levels of Mms4 was quantified. Cell cycle progression of the cells during the experiment was followed by flow cytometric analysis. 1N and 2N in gray indicate G1 and G2/M phases, respectively. Cdc5 levels were detected using anti-Cdc5 antibody. b Time course experiment analyzing the Mms4 species within one cell cycle in mms1Δ cells. Experimental set-up as described in a. c Ni-pulldown of ubiquitylated species of Mms4. His-tagged ubiquitylated proteins (^HisUbi) were immunoprecipitated from cell lysates using Ni-NTA beads. Proteasome-deficient cim3-1 cells of the indicated genotype (WT, esc2Δ, slx5Δ, cul8Δ) expressing HA-tagged Mms4 were grown in Galactose-containing media to induce the expression of His-tagged ubiquitin. HA-Mms4 cells without His-tagged Ubiquitin (no Ubi) and His-tagged Ubiquitin cells without HA-Mms4 (no HA) were used as controls. Protein samples were analyzed by western blot using an anti-Ubiquitin antibody to detect ubiquitylated proteins and anti-HA to detect unphosphorylated Mms4 (^HAMms4), phosphorylated Mms4 (^HAMms4-P), and ubiquitylated Mms4 (^HAMms4-Ubi). Pgk1 and Ponceau staining served as loading controls. Source data are provided as a Source data file.