Modulation of yeast F-actin structure by a mutation in the nucleotide-binding cleft.

Although the actin sequence is very highly conserved across evolution, tissue-specific expression of different isoforms in high eukaryotes suggests that different isoforms carry out different functions. However, little information exists about either the differences in filaments made from different actins or the effects on filament structure caused by the various mutations in actin that have been introduced to gain insight into actin function. Using electron microscopy and three-dimensional reconstruction, we have studied the differences in the filaments made by yeast and rabbit skeletal muscle actin, two proteins with 88% homologous sequences, and we have assessed the changes in filament structure caused by the introduction of the S14A mutation into yeast actin. Elimination of the S14 hydroxyl group, assumed to bind to the gamma-phosphate of actin-bound ATP, results in a 40 to 60-fold decrease in actin's affinity for ATP. We show that yeast actin displays less extensive contacts between the two long-pitch helical strands than does muscle actin, and displays the large cooperativity within filaments previously observed for muscle actin. Finally, we demonstrate that the S14A mutation narrows the cleft between the two lobes of the actin subunit and strengthens the inter-strand connections in F-actin.