Fig. 4: NAC facilitates eEF1A co-translational folding by recruiting Ypl225w to ribosomes.
From: A ribosome-associating chaperone mediates GTP-driven vectorial folding of nascent eEF1A
a ColabFold models of eEF1A DI (light blue) bound to Ypl225w (light purple) and NAC (Egd1: light green, Egd2: dark green). Model on the right was rotated ~50° and highlights the interaction of the ubiquitin-associated (UBA) domain of Egd2 (outlined in gold) with Ypl225w. b Extracts derived from the untagged strain (EGD2, YPL225W) or those tagged at the indicated endogenous loci (EGD2-13xMyc, YPL225W-3xFLAG) were subjected to FLAG IP. Elutions were resolved by SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. The data shown is representative of two independently performed experiments. c The indicated proteins were prepared and subject to FLAG IP as described in “Methods”. Total and eluted (IP) fractions were analyzed by SDS-PAGE and Coomassie staining or immunoblotting (IB) for the Myc tag on Egd2. Ypl225w-3xFLAG was quantitatively captured under these conditions. Dashes indicate cropping from the same gel to remove irrelevant lanes. NAC refers to recombinant Myc-Egd2•10xHis-Egd1. The data shown is representative of two independently performed experiments. d FLAG IP of the indicated proteins was carried out as in panel (c). Dashes indicate cropping from the same Coomassie-stained gel to remove irrelevant lanes. The data shown is representative of two independently performed experiments. e Trypsin resistance of 35S-methionine-radiolabeled eEF1A (45-min in vitro translation) in wild-type (EGD2) or egd2∆ extracts and addition of 100 µg/mL cycloheximide (CHX) to terminate translation. f Trypsin resistance (5-min trypsin digestion time) of 35S-methionine-radiolabeled eEF1A produced from IVT reactions (45-min translation time) in EGD2 or egd2Δ extracts supplemented with the indicated concentrations of recombinant NAC proteins. Dashes indicate cropping from the same gel to remove irrelevant lanes. The data shown is representative of two independently performed experiments. g Trypsin resistance of 35S-methionine-radiolabeled eEF1A was assessed as in panel (f) but with egd2Δ extracts supplemented with the indicated concentrations of Ypl225w-3xFLAG. The data shown is representative of two independently performed experiments.
