A MUTATION IN AN ATP-BINDING LOOP OF SACCHAROMYCES-CEREVISIAE ACTIN (S14A) CAUSES A TEMPERATURE-SENSITIVE PHENOTYPE IN-VIVO AND IN-VITRO

The Ser(14) hydroxyl group of actin is one of six groups that potentia lly form hydrogen bonds with the gamma-phosphate of the ATP bound in t he cleft separating the two domains of the protein, To understand the importance of this group in actin function, we mutated Ser(14) of Sacc haromyces cerevisiae actin and studied the effects of these mutations in vivo and in vitro. Substitution of Cys or Gly resulted in cell deat h. Substitution of Thr for Ser resulted in an actin with wild type pro perties in, vivo and in vitro. Cells carrying the Ser(14) --> Ala (S14 A) mutation were viable but displayed a temperature sensitive lethalit y at 37 degrees C preceded by delocalization of actin patches, the app earance of bar-like structures, and finally the disappearance of ident ifiable actin structures, The mutation caused no effect on the critica l concentration of polymerization but resulted in an actin with an inc reased rate of polymerization, an altered protease susceptibility, and a decreased filament ATPase activity, At 37 degrees C, Mg-, but not C a-S14A-actin irreversibly lost the ability to polymerize, These result s demonstrate the importance of the ATP-Ser(14) hydroxyl hydrogen bond in regulating actin function in, vivo and in vitro and the magnificat ion of the effects of the mutation when Mg(2+)is substituted for Ca2in the protein.