Alb1 is an additional shuttling pre-60S factor and target of Rei1. (A) In the absence of Rei1, Alb1 is blocked in the cytoplasm. The localization of Alb1-GFP was observed in rei1Δ or wild-type strains and grown for 8 h in minimal medium at 23°C. (B) Rei1 is required for the anchoring of Alb1 to pre-60S complexes. rei1Δ or wild-type strains expressing ALB1-GFP were grown for 16 h at 23°C, and whole cell extracts were prepared. The extracts were separated on sucrose gradients and analyzed as described in Fig. 1 C. The immunoblots, performed using antibodies against GFP or Arx1, are displayed below the corresponding profile. (C) Arx1 and Alb1 form a small nonribosomal complex in the absence of Rei1. Arx1- and Alb1-TAP–associated complexes were purified in wild-type or rei1Δ strains grown at 23°C. The Coomassie staining is shown on the left; the right panel displays the results of immunoblots using antibodies against Arx1, Nog1, Rei1, and Tif6 against the TEV eluates. Protein amounts were adjusted relative to Arx1. Positions of the tagged proteins are indicated by asterisks. (D) Arx1 and Kap121 cosediment with Alb1-TAP in rei1Δ strains. The TEV eluate from a purification of Alb1-TAP in the absence of Rei1 was further separated on sucrose gradients either directly (left) or after preincubation with calmodulin affinity resin (right; Alb1 complex subtracted). The components of the fractions were analyzed by immunoblotting using Arx1 and Kap121 antibodies after membrane staining by Ponceau red.