Table II. Summary of Arp2 and Arp3 mutant biochemical and cellular phenotypes.
| Allele
|
Growth
|
ATP cross-linking
|
Etheno-ATP
|
Actin polarization
|
LY uptake
|
Abp1 motility (lifetime) |
Sla1 motility (lifetime) |
Nucleation activity
|
|---|---|---|---|---|---|---|---|---|
| % | Kd (μM) | % | % | % | % | |||
| WT | + | 100 | 2.3 ± 0.1 | 98 (90) | 97 | 94.5 ± 2.1 (15 ± 3 s) |
85.4 ± 5.1 (33 ± 10 s) |
1.0 |
| arp2-D11A | ss | 82.8 | 2.0 ± 0.1 | 72 (59) | 57 | 21.5 ± 5.4 | 9.2 ± 4.4 | 0.11 |
| arp2-G302Y | + | 16.9 | 1.8 ± 0.1 | 98 (90) | 91 | 75.9 ± 8.2 (24 ± 5 s) |
51.5 ± 1.3 (69 ± 24 s) |
0.28 |
| arp2-Y306A | + | 23.1 | 2.4 ± 0.2 | nd | 97 | 94.3 ± 3.4 | 89.1 ± 3.5 (33 ± 8 s) |
1.12 |
| arp3-D11A | ts, fs, ss | 68.1 | 34.5 ± 2.2 | 43 (7) | 14 | 14.5 ± 9.4 | 3.3 ± 2.9 | 0.28 |
| arp3-G302Y | ts, fs, ss | 17.1 | 22.0 ± 4.4 | 18 (4) | 15 | 6.2 ± 4.9 (29 ± 6 s) |
1.7 ± 2.9 (148 ± 52 s) |
0.24 |
| arp3-F306A | + | 29.5 | 16.7 ± 4.2 | nd | 88 | 94.3 ± 3.0 | 78.2 ± 1.7 | 0.96 |
| arp3-Y306A; arp3-G302Y | ss | nd | 1.5 × 106 | nd | 85 | 21.8 ± 1.7 (29 ± 6 s) |
11.0 ± 9.3 (98 ± 27 s) |
0.21 |
Characterization of Arp2 and Arp3 nucleotide-binding mutants. Growth: growth phenotypes on different media. ts, temperature sensitive (37°C); ss, salt sensitive; fs, formamide sensitive. ATP cross-linking: % of wild-type signal in the absence of WCA on Arp2 for arp2 mutants and Arp3 for arp3 mutants. Etheno-ATP K d: average K d ± SEM calculated from data in Fig. 2 (C and D) as described in Materials and methods. Actin polarization: calculated as % of small-budded cells containing <5 patches in the rear of the mother cell at 25°C and 37°C (parentheses). Actin was visualized with rhodamine phalloidin. LY (Lucifer yellow) uptake: measured visually as the % of the cells exhibiting bright vacuolar staining (Fig. 3 C, WT) versus faint or lack of staining (Fig. 3 C, arp3-G302Y). Abp1 motility and Sla1 motility: average % of motile patches ± SD as determined from at least 60 patches for each strain. Parentheses: average lifespan ± SD of patches in seconds as determined from at least 20 patches for each strain. Nucleation activity: fraction of barbed ends generated relative to wild type. nd, not determined.