Fig 5. Analysis of the effect of eEF2 overexpression on the phenotypic defects of the mutants eIF5A^K56A and eIF5A^Q22H/L93F at the permissive temperature (25°C).

(A) Ten-fold serial dilutions of wt, eIF5A^Q22H/L93F or eIF5A^K56A, containing empty vector or high-copy eEF2, were grown in SC-ura at 25°C in the presence or not of sordarin (200 ng/mL). (B) Total protein synthesis was measured in the same cells by the incorporation of [³H]leucine, relative to the wt strain. (C) and (D) Polysome profile analysis of the same cells was carried out and the quantification of polysome and monosome areas (P/M) were performed to show the levels of translation. (E) Polysome profile fractions of eIF5A^Q22H/L93F and eIF5A^K56A in the presence of high-copy eEF2 were analyzed by western blot to detect the presence of eIF5A, eEF2 and Rpl5. The error bars in B and D represent the standard deviation of the experimental values for three independent replicates and the data are expressed as mean values ±SD.