25S rRNA depurination reduces translation and binding of eEF2 to ribosomes. (A) WT BY4741 yeast cells transformed with PAP, PAPx, and EV cDNAs were induced with 2% galactose for 5 h. The methionine analog AHA (L-azidohomoalanine; 50 µM) was then added to cultures for 1 h. Protein synthesis was measured by incorporation of AHA into newly synthesized proteins. Following the covalent binding of a fluorescent alkyne with AHA, incorporated labeled proteins were quantified by fluorescence. Fluorescence values were normalized to OD600 of each sample, and AHA % incorporation values for PAP and PAPx are shown relative to EV values. Bars represent means ± SEM of three independent biological replicates (n = 3). The different letters on bars represent statistically significant differences at P < 0.05. (B) Association of eEF2 with ribosomes of cells expressing PAP, PAPx, or EV was assessed by immunoblot. Total protein and ribosomes were isolated from WT BY4741 cells transformed with cDNAs of PAP, PAPx, or EV following induction with 2% galactose for 6 h. Total cell lysate and ribosomes (20 µg) were separated through 12% SDS PAGE, transferred to nitrocellulose, and probed with eEF2 (1:10,000) and RpL3 (1:5000) antibodies. Immunoblot illustrates eEF2 (93 kDa) and L3 (43 kDa) in total cell lysates and ribosomes.
