βSubunit of Rat Liver Mitochondrial Atp Synthase: Cdna Cloning, Amino Acid Sequence, Expression In Escherichia Coli, and Structural Relationship To Adenylate Kinase

The amino acid sequence of all but a few N-terminal residues of the β subunit of rat liver ATP synthase has been determined from cDNA clones. Rat liver F₁-β is shown to contain 17 amino acid differences from that reported for F₁-β of bovine heart, 2 differences of which involve differences in charge. This may account in part for the observation that bovine heart F₁binds nucleotides with much greater affinity than the rat liver enzyme. Rat liver F₁-β also contains homologous regions with another nucleotide binding protein, adenylate kinase, for which high-resolution structural studies are available. Adjacent to one of these homologous regions is an eight amino acid stretch which bears striking homology to the phosphorylation region of the (Na⁺,K⁺)-ATPase. The combination of these two homology regions may constitute at least part of a nucleotide binding domain in F₁-β. Significantly, both rat liver and bovine heart β contain these regions of homology, whereas the 17 amino acid differences between the two enzymes lie outside this region. The possibility of a second nucleotide binding domain which differs between the two enzymes is discussed. A cDNA clone containing all the regions of homology as well as 11 of the 17 amino acid differences between the bovine heart and rat liver β subunits has been ligated into the bacterial expression vector pKK223-3. After transformation of a protease-deficient strain of Escherichia coli, this cDNA clone is expressed as a 36-kilodalton protein. Finally, further cDNA library screening and primer extension analysis using several oligonucleotide probes generated cDNAs which always terminated prior to that region of the β gene coding for the N-terminal region. A strong secondary structure of regulatory significance may, therefore, be a unique characteristic of the rat liver mRNA coding for the F₁-β subunit in this region.