hRio2 kinase activity is required for the recycling of hDim2, hLtv1, and hNob1 but not hEnp1 from cytoplasmic 40S precursors. (A) Mutation of Lys123 and Asp246 of hRio2 to Ala results in a KD hRio2 mutant. zz-tagged hRio2(WT) and hRio2(K123A,D246A) (hRio2[KD]) were tested for autophosphorylation activity as described in Materials and methods. (B) In hRio2-depleted cells, KD hRio2 rescues the relocalization to the cytoplasm of hEnp1 but not hDim2. Rescue experiments in HeLaK cells were performed as described in Fig. 4 B using EGFP-hRio2(WT) and EGFP-hRio2(KD) as rescue constructs. (C) Western blot analysis of A revealing efficient hRio2 knockdown by RNAi and similar expression levels of the rescue constructs. (D) Quantification of the experiment shown in A. Cells displaying predominantly nucleolar, intermediate, or partially cytoplasmic hEnp1 or hDim2 localization were counted. For rescues, hEnp1 and hDim2 localization was analyzed for transfected cells only. (E and F) Slower shuttling kinetics of hLtv1 and hNob1 after hRio2 depletion are rescued by WT but not KD hRio2. Rescue experiments were performed as described in B and combined with treatment with 20 nM LMB for 1 h as described in Fig. 5 C. Cells were analyzed by immunostaining for hLtv1 (E) and hNob1 (F) and counted as in Fig. 4 C. For rescues, only transfected cells were counted. n, number of cells counted (from one representative experiment); si, siRNA. Bars, 20 µm.