Fig. 1: Mms4 undergoes proteasome-dependent turnover in mitosis.

a Time course experiment analyzing Tc-HA-Mms4 protein levels and stability. Logarithmically (log) grown Tc-HA-Mms4 (WT) cells were synchronized in G1 phase with α-factor (αF) and then released in YPD medium in the absence (−Tet) or presence of 1 mM Tetracycline (+Tet). Additionally, after cells reached G2/M (45 min from the initial release), 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) and phosphorylated Mms4 (^HAMms4-P) species was analyzed by western blot. Cdc5, peaking with Mms4-P in G2/M, was detected using an anti-Cdc5 antibody. Pgk1 served as a loading control. Total levels of Mms4 were quantified by normalization to the loading control and are shown relative to the G1 phase sample. Additionally, the percentage of phosphorylated Mms4 versus total Mms4 is 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. b Time course experiment analyzing the turnover of Mms4 in the presence of the proteasome inhibitor MG132. Similar set-up as in a using pdr5Δ cells to facilitate the uptake of MG132. At 60 min after initial release from G1 arrest, 100 µM MG132 (highlighted in red) or only DMSO (ctrl) was added to the cells, and samples were taken at the indicated timepoints. c Time course experiment analyzing Mms4 levels within one cell cycle in the proteasome mutant cim3-1. Logarithmically grown cells of the indicated genotype were synchronized in G1 and then released in YPD medium containing 1 mM Tetracycline (Tet) at 30 °C. After cells reached G2/M, α-factor was added to arrest cells in the next G1 phase. Samples for western blot and flow cytometric analysis were taken at the indicated timepoints and processed as in a. Source data are provided as a Source data file.