FIGURE 5.

Merging cytoplasmic and mitochondrial ValRS activities into a single gene. CW1 was transformed with constructs encoding E. coli ValRS and its fusions, and the ability of these constructs to rescue the cytoplasmic and mitochondrial defects of the knock-out strain was tested. A, schematic summary of constructs and their complementation activities. Cyt, cytoplasmic; Mit, mitochondrial. B, complementation assays for cytoplasmic ValRS activity on a 5-FOA plate. C, complementation assays for mitochondrial ValRS activity on a YPG plate. D, assay of protein expression by Western blotting. Upper panel, ValRS; lower panel, phosphoglycerate kinase (PGK) (as a loading control). Numbers 1–4 (circled) in B–D represent the constructs shown in A.