Fig. 4.

The conserved Rea1-MIDAS element II loop is required for Rea1’s essential function. a The indicated constructs were transformed in a rea1∆ shuffle strain and the viability of the respective mutants assessed on 5-FOA-containing plates. Cells were grown at 30 °C for 3 (SDC-Leu) and 5 days (SDC + FOA). b The indicated constructs under control of the GAL1-10 promoter were transformed in a wild-type strain (expressing endogenous REA1) and cell growth was monitored after incubation at 30 °C on plates containing glucose (SDC-Leu, left) or galactose (SGC-Leu, right) for 2 and 3 days, respectively. c Affinity purification of the indicated Rea1 constructs fused to an N-terminal TAP-Flag tag and under control of the endogenous REA1 promoter. The constructs were transformed into an AID-HA-REA1 degron strain. The endogenous Rea1 was depleted by the addition of auxin (final concentration 500 µM) to the cultures 2 h before cells were harvested. Final eluates were analyzed by SDS-PAGE and Coomassie staining or by western blot analysis against pre-60S assembly factors and ribosomal protein L3, using the indicated antibodies. d The subcellular localization of the indicated full-length Rea1 constructs N-terminally fused to GFP was monitored by fluorescence microscopy. Scale bar: 5 µm. Source data are provided as a Source Data file