Fig. 1: The Get4/5 complex binds to ribosomes with high affinity.

a The Get4/5 complex binds to ribosomes via Get5. Total cell extracts (tot) of wild type, a Δget4 strain overexpressing Get5 (Δget4 + Get5↑, Supplementary Fig. 1), or a Δget5 strain were separated into a cytosolic fraction (cyt) and a ribosomal pellet (rib) under low salt (120 mM KOAc) or high salt (800 mM KOAc) conditions. The black asterisk indicates the fraction of overexpressed Get5 migrating with lower molecular mass, likely due to partial degradation of overexpressed Get5. The white asterisk indicates the fraction of aggregated Get4, which sediments together with ribosomes under high salt conditions in the absence of Get5. Aliquots were analyzed via immunoblotting with α-Get4, α-Get5, α-Rpl35 (ribosomal marker), and α-Pgk1 (cytosolic marker). b Quantification of the fraction of Get4 and Get5 bound to ribosomes. Given is the fraction of Get4 or Get5 recovered in low salt ribosomal pellets (rib) compared with the total (rib+cyt) based on the analysis of experiments as shown in a. Get4 or Get5 recovered in high salt ribosomal pellets was subtracted as a background. The results of each biological replicate are shown as black dots. Error bars indicate the standard deviation of the mean. Indicated p values were calculated using a two-sided Student’s t test with GraphPad Prism. c Get4/5 binds to purified 80S ribosomes with high affinity. Fluorescence anisotropy-based binding assays were performed with Get4/5-Atto390 (20 nM) and purified 80S ribosomes (see Methods). The data were fit to a one site, saturation binding curve, from which the equilibrium dissociation constant and the standard error of the mean (K_d = 110 ± 40 nM) was determined using GraphPad Prism. d Get4/5 binding to the 80S ribosome monitored by FCS. Autocorrelation functions are represented for Get4/5-Atto655 alone (red) and in complex with the 80S ribosomes (blue). Concentrations of Get4/5-Atto655 and 80S ribosomes were 5 nM and 1 µM, respectively.