FIGURE 2.

Converting E. coli ValRS into a functional yeast cytoplasmic enzyme. CW1, a yeast VAS1 knock-out strain, was transformed with constructs encoding various ValRSs, and the ability of these constructs to rescue the cytoplasmic defect of the knock-out strain was tested. A, schematic summary of constructs and their complementation activities. The symbols + and − indicate positive and negative complementation, respectively. Cyt, cytoplasmic. B, complementation assays for cytoplasmic ValRS activity on a 5-FOA 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.