BERYLLIUM FLUORIDE AND PHALLOIDIN RESTORE POLYMERIZABILITY OF A MUTANT YEAST ACTIN (V266G,L267G) WITH SEVERELY DECREASED HYDROPHOBICITY IN A SUBDOMAIN 3 4-LOOP/

Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3 /4 loop interact with a hydrophobic pocket on the opposing strand resu lting in helix stabilization, We have determined how a decreased hydro phobicity of this plug affects yeast actin function. Cells harboring o nly the V266G, V266D, V266F, L267G, L269D, or L269K actins appear norm al, 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 G G cells, V266D actin polymerizes slower than wild-type actin at room t emperature, At 4 degrees C, not only is polymerization slowed, but the re is also an effect on critical concentration, However, the polymeriz ation defects are milder than those associated with substitution of As p 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, b eryllium fluoride, or phalloidin at room temperature, although at 4 de grees C only phalloidin is effective, These results suggest that the d iminished hydrophobicity of the plug in GG actin leads to filament des tabilization. However, the V266D actin results require a modification of the original Holmes filament model.