mCherry fluorescent reporter for mistranslation. One of the three amino acid residues that form the fluorophore in mCherry is Met72. (A) mCherry lost fluorescence in E. coli when Met72 was mutated to Lys (AAA, AAG), Asp (GAC, GAT), and Glu (GAG, GAA), as determined by flow cytometry (AAG mutant shown). All mutant proteins in E. coli are as stable as the WT protein as shown by Coomassie brilliant blue stain of the total cell protein (bottom). (B) EGFP and WT mCherry transiently transfected to HeLa cells. Transfection was done either using the fusion protein (left) or just the EGFP or mCherry alone, showing little signal overlap between these two proteins. Y-axis on the left (bar graphs) shows EGFP and mCherry at the same intensity; y-axis on the right (circles) shows the amount of plasmids used in transfection. (C) EGFP–mCherry dual fluorescence reporter mutants were transiently transfected to HeLa cells and cells were analyzed by flow cytometry. Cells transfected with the reporter harboring a Glu mutation show high residual mCherry fluorescence. Same amounts of plasmids as in panel B were used in transfection. Y-axis on the left (green bars) shows eGFP intensity; y-axis on the right (red bars) shows mCherry intensity. Note that the left and right y-axes have different scales.
