TABLE 1:
Frequency of CTP synthase assembly, fractions of foci and filaments, average and median lengths of structures, and total number of structures used for analysis.
| Mutants | Description | Cells with structures (%) | Percentage foci | Percentage filaments | Average length of structures (μm) | Median length of structures (μm) | Number of structures analyzed |
|---|---|---|---|---|---|---|---|
| Wild type | 26.03 ± 1.72 | 30.63 ± 3.24 | 69.37 ± 3.24 | 1.053 ± 0.039 | 1.059 | 620 | |
| E161K | CTP-binding site | 99.86 ± 0.09 | 98.47 ± 0.59 | 1.53 ± 0.59 | 0.363 ± 0.018 | 0.359 | 784 |
| E161K-C404G | CTP-binding site and catalytic site | 64.31 ± 4.43 | 99.41 ± 0.30 | 0.59 ± 0.30 | 0.355 ± 0.007 | 0.347 | 322 |
| E146A | ATP-binding site | 56.91 ± 3.98 | 26.99 ± 3.46 | 73.01 ± 3.46 | 1.171 ± 0.027 | 1.233 | 431 |
| D70A | ATP-binding site | 54.69 ± 4.08 | 36.40 ± 4.24 | 63.60 ± 4.24 | 0.893 ± 0.048 | 0.895 | 402 |
| R381M | GTP-binding site | 86.37 ± 2.69 | 13.90 ± 3.13 | 86.10 ± 3.13 | 1.370 ± 0.036 | 1.407 | 415 |
| R381P | GTP-binding site | 81.91 ± 6.61 | 17.92 ± 1.59 | 82.08 ± 1.59 | 1.202 ± 0.022 | 1.216 | 388 |
| G382A | GTP-binding site | 58.16 ± 4.80 | 46.00 ± 9.86 | 54.00 ± 9.86 | 0.828 ± 0.067 | 0.789 | 480 |
| C404G | Catalytic-site | 29.13 ± 2.57 | 29.11 ± 0.93 | 70.89 ± 0.93 | 1.030 ± 0.016 | 1.043 | 385 |
| G148A | Tetramerization | 72.08 ± 9.90 | 27.80 ± 3.36 | 72.20 ± 3.36 | 1.150 ± 0.018 | 1.179 | 519 |
| S330A | Phosphorylation site | 28.18 ± 1.86 | 39.86 ± 3.50 | 60.14 ± 3.50 | 0.905 ± 0.042 | 0.887 | 315 |
| S354A | Phosphorylation site | 27.77 ± 2.70 | 46.20 ± 2.39 | 53.80 ± 2.39 | 0.806 ± 0.027 | 0.778 | 431 |
| S424A | Phosphorylation site | 24.33 ± 3.57 | 36.50 ± 3.54 | 63.50 ± 3.54 | 0.911 ± 0.040 | 0.887 | 404 |
| S36A | Phosphorylation site | 13.90 ± 3.92 | 36.20 ± 0.16 | 63.80 ± 0.16 | 0.913 ± 0.031 | 0.905 | 395 |
| S36D | Phosphorylation site | 6.084 + 1.04 | N/A | N/A | N/A | N/A | N/A |
| S36E | Phosphorylation site | 5.346 ± 1.37 | N/A | N/A | N/A | N/A | N/A |
Percentage of cells with structures was collected by counting 250–300 cells grown for 1 d in YPD and fixed with 3.36% formaldehyde. The experiments were repeated five times, and the average ± SEM was calculated. Percentage foci and percentage filaments were obtained by imaging cells grown for 1 d in YPD and fixed with 3.36% formaldehyde with the DeltaVision system. Imaging was done for three independent repeats (except for wild type, six repeats). Deconvolved and compressed images were analyzed by Fiji, and the structures with length <0.75 μm were defined as foci and those with length ≥0.75 μm as filaments.