ATP synthase from bovine mitochondria: The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs

Abstract

ATP synthase from bovine mitochondria is a complex of 13 different polypeptides, whereas the Escherichia coli enzyme is simpler and contains eight subunits only. Two of the bovine subunits, b and d, which had not been characterized, have been isolated from the purified enzyme. Subunits with sizes corresponding to bovine subunits b and d are evident in preparations of the enzyme from mitochondria of other species. Partial protein sequences have been determined by direct methods. On the basis of some of this information, two oligonucleotide mixtures, 17 and 18 bases in length, have been synthesized and used as hybridization probes in the isolation of clones of the cognate cDNAs. The sequences of the two proteins have been deduced from their DNA sequences. Subunit b is 214 amino acid residues in length and has a free N terminus. Subunit d is 160 amino acid residues long. Its N-terminal alanine is blocked by an N-acetyl group, as demonstrated by fast atom bombardment mass spectrometry of N-terminal peptides. The sequence near the N terminus of the b subunit is made predominantly of hydrophobic residues, whereas the remainder of the protein is mainly hydrophilic. This N-terminal hydrophobic region may be folded into an β-helical structure spanning the lipid bilayer. In its distribution of hydrophobic residues, this protein resembles the b subunits of ATP synthase complexes in bacteria and chloroplasts. The b subunit in E. coli forms an important structural link between the extramembrane sector of the enzyme F 1, and the intrinsic membrane domain, F 0. It is proposed that the bovine mitochondrial subunit b serves a similar function. If this is so, the mitochondrial enzyme, as the chloroplast ATP synthase, contains equivalent subunits to all eight of those that constitute the E. coli enzyme. Subunit d has no extensive hydrophobic sequences, and is not apparently related to any subunit described in the simpler ATP synthases in bacteria and chloroplasts.