Yeast Whole-Cell Extract Preparation

Yeast whole-cell extracts were prepared as described previously64 (link). In brief, cells at OD₆₀₀ of 0.5 in log-phase were collected and washed immediately with 20% trichloroacetic acid (TCA). The cell pellet was suspended in 250 μl of 20% TCA, mixed with an equal volume of glass beads (Sigma, 425–600 μm) and vortexed for 15 min at 4 °C. glass beads were washed twice with 250 μl of 5% TCA to increase the protein yield. The resulting extract was spun at 13,000 r.p.m. for 5 min at 4 °C. The pellet was dissolved in 100 μl of 2 × SDS-loading buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.1% bromophenol blue, 50 mM DTT), neutralized by adding 20 μl of 1 M Tris base, boiled for 5 min and clarified by centrifugation. Total cell extracts were subjected to immunoblot analysis. Proteins were detected using the following primary antibodies: mouse anti-Pgk1 (1:10,000; Invitrogen, 459250), rabbit anti-Clb2 (1:400; Santa Cruz Biotechnology, y-180), rabbit anti-Cof1 (1:2,000), mouse anti-Actin (1:1,000, Thermo Fisher scientific, mAbGEa) and rabbit anti-Sac6 (1:2,000). Blots were subsequently scanned using Odyssey Infrared Imager (LI-COR Biosciences). Full scans are provided in Supplementary Fig. 12.