Loss of enzymatic activity is not common to DI-CMTC associated TyrRS mutations. (A–C) TyrRS and dTyrRS are functional homologs. Scabrous-GAL4-driven expression of dYARS-RNAi induced bristle phenotypes: scutellar bristles (arrows in A) were often shorter or missing, with the dorsal scutellar bristles displaying more severe defects than the anterior scutellar bristles (arrowheads in B). This was never the case in genetic controls (A). YARS_WT coexpression (C) resulted in full rescue of scabrous-GAL4 > dYARS-RNAi induced bristle phenotypes. (D–F) dYARS-RNAi induced bristle phenotypes could also be fully rescued by coexpression of YARS_E196K (D), but not by YARS_G41R (E) and YARS_153-156delVKQV (F). (G) Genetic complementation experiments in S. cerevisiae. Single copy plasmids encoding WT or mutant TYS1 (the S. cerevisiae YARS ortholog) under the regulation of its native promoter and terminator were transformed into a hemizygous TYS1-deletion strain. Transformants were sporulated and resulting tetrads were dissected to obtain haploid spores. Viability of 50% of the spores depends on the aminoacylation activity of the transgene. Segregant colonies expressing TYS1_WT or TYS1_E196K had normal colony size, consistent with normal aminoacylation activity. TYS1_153-156delVKQV colonies were viable but displayed reduced colony size and TYS1_G41R colonies were nonviable, indicative for severe reduction of aminoacylation activity. (H) Enzymatic activity of recombinant full-length WT and mutant TyrRS proteins determined in an in vitro aminoacylation assay (14).

Expression of mutant YARS in Drosophila induces impaired motor performance, electrophysiological evidence of neuronal dysfunction, and defects in axon terminal morphology. (A) Impaired performance of mutant YARS flies in a negative geotaxis climbing assay. The average time needed to climb a vertical wall to a height of 82 mm is displayed. Actin5C-GAL4^weak was used to drive 1 copy of transgene; actin5C-GAL4^strong was used to drive 2 copies of transgene. Control flies carry transgenes without driver. (B and C) Flight (B) and jump (C) ability of actin5C-GAL4^weak > YARS_E196K flies progressively declines with age. In a flight assay (16), the percentage of flies that fell in a straight line, fell by veering or flew was determined at the indicated ages (B). The same flies were tested for jumping ability (16) (C). All genetic control flies were able to fly and jump at all ages tested (see Table S3). (D) Specific expression of mutant YARS in neurons induced impaired motor performance in a negative geotaxis assay. Nsyb-GAL4 was used to drive 2 copies of YARS transgenes, flies lacking the driver were used as controls. (E) Negative geotaxis assay on nsyb-GAL4 > 2× dYARS flies revealed impaired motor performance in flies expressing mutant—but not WT—dYARS panneuronally. (F) Electrophysiological defects in the giant fiber system of mutant YARS expressing flies. The percentages of flies without electrophysiological synaptic defects are shown for each age group. More than 20 female flies carrying A307-GAL4 and 2 copies of YARS transgene were tested for each age group. A functional defect is defined as a response latency > 1 ms and/or the inability to follow 100-Hz stimuli one-to-one. All comparisons of mutant YARS versus YARS_WT are statistically significant, except for the 1-day time point where 2×E196K and 2×G41R are not significantly different from WT. (G) To determine the correlation between GF function and morphology, dye-filling experiments were performed of electrophysiologically tested flies, which revealed the morphology of the giant fiber. In flies with no physiological phenotype or with defects only in following stimuli at 100 Hz, no gross anatomical defects were seen. In 100% of flies with increased response latency or no TTM response upon brain stimulation, the giant synaptic terminal was abnormally thin with occasional vesicles (arrowhead) or exhibited constrictions (arrow). A307-GAL4 was used to drive expression of the indicated transgenes. ***, P < 0.001; indicates significant differences versus genetic controls, as determined by one-way ANOVA with Bonferroni's Multiple Comparison Test.