Drg1 specifically releases Rlp24 from pre-60S particles. (A) Pre-60S particles from the temperature-sensitive drg1-18 mutant incubated at 37°C were purified with Arx1-TAP as bait and immobilized on calmodulin beads. After incubation with Drg1 in the presence (+) or absence (−) of ATP and the Nup116 fragment N172, supernatants (containing released and unbound proteins) were collected and TCA precipitated. pre-60S particles were eluted from the calmodulin beads and concentrated by TCA precipitation. Supernatants (released) and eluates (pre-60S bound) were analyzed by SDS-PAGE and Coomassie staining, as well as Western blotting. (B) Pre-60S particles purified from the Δnup116 as well as from the nup116Δ110-166 mutant show increased amounts of Drg1 and Rlp24. The nup116Δ strain carrying centromeric plasmids with wild-type NUP116 or the nup116 variant lacking codons 110–166 were grown to late log phase and pre-60S particles were isolated via Arx1-TAP. Purified particles were separated by SDS-PAGE and analyzed for the levels of pre-60S maturation factors by Western blotting.