Fig. 2: Ypl225w is a bona fide eEF1A co-translational chaperone.

a Trypsin resistance of ³⁵S-methionine-radiolabeled eEF1A was determined following in vitro translation (IVT) for 45 min in wild-type (YPL225W) or ypl225w∆ extracts and addition of 100 µg/mL cycloheximide (CHX) to terminate further protein synthesis. The “0” time point represents the undigested sample. Following digestion for the indicated times, samples were resolved by SDS-PAGE and visualized by autoradiography. “Fraction resistant” is the signal of near full-length, trypsin-resistant (Tryp.-Res.) eEF1A fragment divided by the total synthesized eEF1A in the undigested “0” time point. Samples were also analyzed by immunoblotting (IB) with the indicated antibody. The data shown is representative of three independently performed experiments. b Trypsin resistance of ³⁵S-methionine-radiolabeled eEF1A (45-min translation time) was assessed after 5 min of trypsin digestion and with addition of the indicated amounts of recombinant Ypl225w-3xFLAG protein prior to IVT. Samples were also analyzed by IB with the indicated antibodies. “Fraction resistant” is the signal of near full-length, trypsin-resistant (Tryp.-Res.) eEF1A fragment divided by the total synthesized eEF1A in the corresponding undigested lane normalized to the WT YPL225W signal. The data shown is representative of three independently performed experiments. c Trypsin resistance of ³⁵S-methionine-radiolabeled eEF1A (20-min translation time) was assessed after 5 min of trypsin digestion. “Co-translational addback” reactions were supplemented with Ypl225w-3xFLAG protein prior to translation initiation and immediately treated with trypsin post-CHX addition. “Post-translational addback” reactions were only supplemented with Ypl225w-3xFLAG protein after CHX treatment and then incubated at room temperature for 20 min before trypsin treatment. The data shown is representative of two independently performed experiments. d Affinity-purified samples from one replicate of the untagged (YPL225W) and YPL225W-3xFLAG samples that were used for mass spectrometry analysis shown here after SDS-PAGE followed by silver staining. The data shown is representative of two independently performed experiments. e Mass spectrometry analysis of samples similar to the ones in panel (d). The dotted line represents a false discovery rate at the p-value of 0.05. The FDR was calculated on the Empirical Bayes moderated t-statistic (see “Methods”). The inset within the volcano plot represents various categories of the enriched Ypl225w-3xFLAG interactors. The black dots represent interactors outside of the indicated categories.