Fig. 4
Rio2 ATP hydrolysis stimulated by the Rps20 loop triggers release of Ltv1. a–c In vitro phosphorylated Ltv1 remains trapped on pre-40S particles containing the catalytically inactive Rio2.D253A or the mutant Rps20Δloop protein. Tsr1-TAP particles from cells expressing wild-type or mutant alleles of LTV1 (left panel), RPS20 (middle panel), and RIO2 (right panel) were immobilized on IgG beads. Particles were incubated in the absence (−) or presence (ATP) of ATP, washed, and eluted with TEV protease. Eluates were analyzed by western blotting using the indicated antibodies. The displayed blots within each section (a–c) originate from the same membrane and the same exposures, allowing for direct comparison of the levels of detected proteins. d The Rps20 loop stimulates Rio2 ATPase activity. Relative ATPase activity obtained from purified Tsr1-TAP particles, carrying the indicated RIO2 or RPS20 alleles, was monitored by single-turnover experiment. The input was adjusted to equal amounts of Rio2 (right panel). An exemplary thin layer chromatography showing ATP and released phosphate (Pi) is depicted (upper left panel). Mean values from quantification of the results obtained from two biological and two technical replicates are plotted (lower panel). Error bars represent standard deviations. Source data are provided as a Source Data file
