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 s uggests 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 i nsight into actin function. Using electron microscopy and three-dimens ional 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 st ructure caused by the introduction of the S14A mutation into yeast act in. Elimination of the S14 hydroxyl group, assumed to bind to the gamm a-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 exte nsive contacts between the two long-pitch helical strands than does mu scle actin, and displays the large cooperativity within filaments prev iously observed for muscle actin. Finally, we demonstrate that the S14 A mutation narrows the cleft between the two lobes of the actin subuni t and strengthens the interstrand connections in F-actin. (C) 1997 Aca demic Press Limited.