Beryllium Fluoride and Phalloidin Restore Polymerizability of a Mutant Yeast Actin (V266G,L267G) with Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop

Abstract

Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization. We have determined how a decreased hydrophobicity of this plug affects yeast actin function. Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern. However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells. V266D actin polymerizes slower than wild-type actin at room temperature. At 4°C, not only is polymerization slowed, but there is also an effect on critical concentration. However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu 267. Purified GG-actin does not polymerize in vitro alone or in the presence of wildtype F-actin seeds. GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4°C only phalloidin is effective. These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization. However, the V266D actin results require a modification of the original Holmes filament model.

Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

“…Thus, it is conceivable that alteration of this hydrophobic residue from L270K may have a profound affect when combined with other changes seen in parasite actins as described above. Importantly, the instability of TgACT1 filaments was partially restored in the presence of phalloidin and JAS, which would be expected to stabilize cross-subunit interactions within the filament, based on previous results with yeast (Kuang and Rubenstein, 1997).…”

“…For example, alteration of L267 to G in yeast actin results in a coldsensitive phenotype characterized by unstable filaments (Chen et al, 1993). Other mutations within this hydrophobic stretch including L269D or L269K had little discernable effect on actin polymerization in vitro (Kuang and Rubenstein, 1997). Although this result might suggest that residues at the C-terminal end of the hydrophobic plug play a less important role, the specific alteration seen in TgACT1 (L/M to K) has not been tested in the context of other alterations seen in parasite actins.…”

Interaction in Vivo and in Vitro between the Yeast Fimbrin, SAC6P, and a Polymerization-defective Yeast Actin (V266G and L267G)

“…However, polymerization can be restored by inclusion of phalloidin or beryllium fluoride (BeFx), 1 although the effects of these agents on GG-actin polymerization differ in a temperature-dependent fashion. Phalloidin will rescue polymerization at temperatures all the way down to 4°C, presumably because it forms an inter-strand interaction (3,4,6), which substitutes for the one disturbed by the GG mutation. BeFx on the other hand, restores polymerization at 25°C but not at 4°C (4).…”

“…Phalloidin will rescue polymerization at temperatures all the way down to 4°C, presumably because it forms an inter-strand interaction (3,4,6), which substitutes for the one disturbed by the GG mutation. BeFx on the other hand, restores polymerization at 25°C but not at 4°C (4). This agent substitutes for the ␥-phosphate of ATP that is released from the interior of the monomer following polymerization and restores the filament to a more stable ADP-P i like state, apparently by strengthening monomer-monomer contacts along the filament axis (7,8).…”

“…Our laboratory previously constructed an actin mutant (GG) in which the first two residues in the subdomain 3/4 loop were replaced by glycines (4,5). The GG mutant cells are coldsensitive and slow growing, and at all temperatures cells contain no visible actin cables and are characterized by delocalized cortical patches.…”