Fig. 3: Ypl225w’s chaperone function is restricted to eEF1A DI.
From: A ribosome-associating chaperone mediates GTP-driven vectorial folding of nascent eEF1A
a WT (YPL225W) and ypl225wΔ strains expressing eEF1A DI-GFP driven by the GAL1 promoter (integrated at the URA3 locus) were imaged by confocal microscopy and analyzed as in Fig. 1d. Scale bar represents 2.5 µm. 100 cells were quantified for each sample (right panel). See “Methods” for details of how expression of GAL1 was induced. The data shown is representative of three independently performed experiments. b Strains expressing eEF1A DI-GFP from panel (a) were analyzed via flow cytometry as described in Fig. 1e. Measurements were compared by an unpaired, two-tailed t-test and yielded a p-value of 0.0000077. ****p < 0.0001. c Cartoon schematic of ribosome-nascent chain complexes (RNCs) and sample processing for RNC-crosslinking experiment in panel (d). V160/V332 represent stalled RNCs of 160 or 332 amino acids in length stalled at a valine (V) codon. Cartoon templates have been used in our previous work11,12. d IVT reactions from ypl225wΔ extracts supplemented with 1 µM Ypl225w-3xFLAG and nonstop eEF1A mRNAs (V160 or V332 RNCs) were translated for 15 min prior to crosslinking with 1 mM BS3. Samples were then subjected to denaturing FLAG immunoprecipitations (IPs) and analyzed by SDS-PAGE followed by autoradiography. The data shown is representative of three independently performed experiments. Filled arrow: V160-NC, asterisk: V160-tRNA, open arrow: V160-tRNA x Ypl225w-3xFLAG, filled circle: V160-NC x Ypl225w-3xFLAG, open square: V332-NC, open circle: V332-tRNA.
