Fig 4.

Functional assays of GRS2 variants. Constructs bearing GRS2 or its derivatives were transformed into a grs1⁻ strain of Saccharomyces cerevisiae, and the ability of the transformants to grow on 5-FOA and YPG media was tested. (A) Summary of the constructs and their rescue activities. Mit, mitochondrial; Cyt, cytoplasmic; MTS, mitochondrial targeting signal; IP, insertion peptide; GRS2(CO), a codon-optimized GRS2. (B) Rescue of cytoplasmic GlyRS activity. (C) Rescue of mitochondrial GlyRS activity. (D) Western blotting. Top, GlyRS; bottom, PGK (as a loading control). (E) Relative protein levels of GlyRS1, GlyRS2, and GlyRS2(CO). (F) Fluorescence microscopy. MitoTracker and 4′,6-diamidino-2-phenylindole (DAPI) were used to label mitochondria and nuclei, respectively. Indicated at the bottom of the Western blots are the amounts of protein extracts loaded into the gels. Numbers 1 to 8 (circled) in panels B to E represent constructs shown in panel A.