Figure 3. Nug2 release from pre-60S particles requires intrinsic K⁺-dependent GTPase and Rea1 ATPase activity.

(a) Synthetic lethality (sl) between alleles rsa4-1²¹ or rea1-DTS²¹ and nug2K328R revealed by growth on 5-FOA. (b-e) ATP-dependent release of Rsa4 and Nug2 from purified pre-60S particles. Scheme of the release assay (b) and experimental analyses (c-e). Affinity-purified Rix1-particles carrying wild-type or mutant Nug2 were incubated with ATP or GTP in NaCl or KCl buffer, before matured pre-60S particles were re-isolated via RpL3-Flag affinity-purification. Final eluates were analyzed by SDS-PAGE and Coomassie staining (upper panel; indicated bands were identified by mass spectrometry) and Western blotting using the indicated antibodies (lower panel). All in vitro assays were performed at least twice with highly reproducible datasets.