Exchangeability of the b subunit of the Cl−-translocating ATPase of Acetabularia acetabulum with the β subunit of E. coli F1-ATPase: Construction of the chimeric β subunits and complementation studies

AbstractThe gene encoding the b subunit of the Cl−-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids). The deduced amino acid sequence showed high similarity to the β subunit of the F type ATPases, but was different in the N-terminal 120 amino acids. The role of the N-terminal region was investigated using an F1-ATPase β-less mutant of E. coli, JP17. The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction. The β subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C). The corresponding regions of ACLB were designated as portions A′ (from 1 to 106), B′ (from 107 to 172) and C′ (from 173 to 478). Chimeric proteins with combinations of A–B′–C′, A–B–C′ and A′–B–C restored the function as the β subunit in E. coli F0F1-complex, but those with combinations of A′–B′–C and A–B′–C had no function as the β subunit. These findings suggested that portion B plays an important role in the assembly and function of the β subunit in the F0F1-complex, while portion B′ of ACLB exhibited inhibitory effects on assembly and function. In addition, portion A was also important for interaction of the β subunit with the α subunit in E. coli F0F1-complex. These findings also suggested that the b subunit of the Cl−-translocating ATPase of A. acetabulum has a different function in the Cl−-translocating ATPase complex, although the primary structure resembled to the β subunit of the F1–ATPase