FIG 1 .

Ablation of K40 acetylation is not tolerated unless it is replaced with the K40Q acetyl-lysine mimic. (A) Diagram of TgTUBA1 genomic locus aligned with the construct used to replace the endogenous locus by double homologous recombination. The construct contains the nonstabilizing V252L oryzalin resistance mutation and the K40K, K40R, or K40Q mutation. (B) IFAs of K40 mutants stained for acetyl-K40–α-tubulin (red) or β-tubulin (specific to Toxoplasma, green). The K40Q mutation results in complete loss of K40 acetylation in the parasite but not its host cell. Images were merged with the DNA stain DAPI (blue). Scale bars, 3 μm. (C) Western blot (WB) assay of parental RH and mutant parasites showing loss of K40 acetylation in K40Q mutants. The blot was probed with anti-acetyl-K40–α-tubulin and anti-SAG1 antibodies as a loading control. (D) Doubling assays performed to assess the growth of parental RH and mutant parasites. Replication rates were determined by counting the parasites within 100 random vacuoles at 24 h postinfection. Three independent trials were conducted, and the average percentage of vacuoles with the indicated number of parasites ± the standard error of the mean is shown (no significant difference between mean percentages of vacuoles at each stage between strains as determined by two-way analysis of variance).