FIGURE 3.

Conversion of E. coli ValRS into a functional yeast mitochondrial enzyme. CW1, a yeast VAS1 knock-out strain, was transformed with constructs encoding various ValRSs, and the ability of these constructs to rescue the mitochondrial defect of the knock-out strain was tested. A, schematic summary of constructs and their complementation activities. Mit, mitochondrial. ScValRS(M1A) specifies only the cytoplasmic form of yeast ValRS due to a mutation at the AUG1 initiator codon. B, complementation assays for mitochondrial ValRS activity on a YPG plate. C, assay of protein expression by Western blotting. Upper panel, ValRS; lower panel, phosphoglycerate kinase (PGK) (as a loading control). Numbers 1–6 (circled) in B and C represent the constructs shown in A.