Gain-of-function eIF6 mutant bypasses the requirement for SBDS in Dictyostelium. (A-B) eIF6^T56K suppressor rescues the growth defect of SBDS-depleted cells. Growth of sbdS^int(ts) cells transformed with empty vector control (red squares) or a plasmid expressing eIF6^T56K (blue diamonds) compared with wild-type (black circles; A) on bacterial lawns at 22°C, where values represent the mean diameter ± SD of 10 plaques from 1 of 3 representative experiments or (B) in axenic medium at 27°C, where values represent mean ± SD of 3 experiments. (C) Comparison of polysome profiles from sbdS^int(ts) cells transformed with empty vector control (pDXA-3C) or a plasmid expressing eIF6^T56K (pDXA-eIF6^T56K). (D) Expression of eIF6^T56K suppressor protein or actin control in sbdS^int(ts) cells, visualized by immunoblotting of cell extracts. (E) SBDS is required for nuclear recycling of eIF6. Subcellular fractionation of wild-type compared with sbdS^int(ts) cells grown at 22°C or 27°C for 12 hours was performed and the indicated proteins visualized by immunoblotting. Histone H3, nuclear marker. Extracts from “total” and “cytoplasmic” fractions represent 2 × 10⁵ cells; nuclear fraction, 2 × 10⁶ cells. (F) Quantification of eIF6 redistribution in SBDS-depleted cells. Ratios were calculated by densitometry. Cyto indicates cytoplasmic; and Nuc, nuclear. (G) eIF6 cosediments with 60S subunits in SBDS-depleted cells. Extracts from wild type control and sbdS^int(ts) cells were fractionated by sucrose gradient sedimentation in high-salt (0.5M KCl) buffer and immunoblotted to detect eIF6. The sedimentation positions of 40S, 60S, and 80S ribosomes are indicated. Input (In) represents 1% of loaded lyzate.