Figure 8.

The activity of OSGEPL1 is influenced by post-translational modification. Higher energy collision-induced dissociation (HCD) MS/MS spectra were recorded for (A) the [M+2H]²⁺ ion at m/z 821.9313 for human OSGEPL1 peptide SLDIAPGDMLDKVAR harbouring one acetylated site (Lys203), and (B) the [M+4H]⁴⁺ ion at m/z 534.5298 for the human OSGEPL1 peptide AADIAATVQHTMACHLVKR harbouring one acetylated site (Lys299). Predicted b- and y-type ions (not all) are listed above and below the peptide sequences, respectively. Matched ions are labelled in the spectra. (C) Genes encoding Kae1, OSGEPL1-ΔMTS, -K74Q, -K140Q, -K203Q, -K230Q, -K240Q and -K299Q were transformed into ScΔKae1, transformants were initially cultured in SD/Leu⁻ liquid medium, spread on SD/Leu⁻ or SD/Leu⁻/5-FOA plates, and the growth phenotype was observed on two plates treated with the same 10-fold diluted concentrations (initial OD₆₀₀ = 1.0) as indicated. Kae1 and p425TEF empty vector were used as positive and negative controls, respectively. (D) Western blotting analysis was performed with yeast extracts before 5-FOA selection. (E) t⁶A modification activities of OSGEPL1-ΔMTS (brown filled circles), K74Q (red filled squares), K203Q (green filled inverted triangles), K230Q (blue filled diamonds), K240Q (violet filled circles) and K299Q (orange filled triangles).