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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
 

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 Kd: average Kd ± 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.

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