δ Subunit of rat liver mitochondrial ATP synthase: Molecular description and novel insights into the nature of its association with the F₁-moiety

The F₁ moiety of ATP synthase complexes consists of five subunit types in the stoichiometric ratio α₃β₃γδε. Of these, the δ subunit has received very little attention in the study of F₁ preparations from eukaryotic cells. Although recently shown to associate tightly with the β subunit [Pedersen, P. L., Hullihen, J., Bianchet, M., Amzel, L. M., and Lebowitz, M. S. (1995) J. Biol. Chem. 270, 1775-1784], the δ subunit is not resolved in the X-ray structure of either the rat liver or bovine heart enzyme. For these reasons, the novel studies reported here were designed both to provide a molecular description of the rat liver δ subunit and to gain insight into the nature of its interaction with F₁. The rat liver δ subunit was cloned from a λgt11 library, sequenced, overexpressed in Escherichia coli (E. coli) in fusion with the maltose binding protein, and, after cleavage of the latter protein, purified to homogeneity. The purified δ subunit (MW = 14.7 kDa) was shown by circular dichroism spectroscopy to be highly structured and to exhibit about 25% sequence identity to the chloroplast and E. coli ε subunits, frequently regarded as homologues of higher eukaryotic δ subunits. Significantly, and in contrast to the chloroplast and E. coli ε subunits, which are readily removed from their parent F₁ moieties after treatment respectively with ethanol and lauryldimethylamine oxide, the rat liver δ subunit remained tightly bound to F₁ under these relatively mild conditions. For the above reasons, four types of experiments were carried out on rat liver F₁ in order to (1) determine the accessibility of the δ subunit to both specific antibodies and to proteases, (2) establish the effect of nucleotides on this subunit's accessibility, (3) identify in cross-linking studies with disuccinimidyl glutarate this subunit's most reactive neighbor, and (4) determine whether this subunit can be dissociated from F₁ by using ionic detergents while leaving the remaining complex intact. The data derived from this detailed set of studies (a) supports the view that the rat liver F₁-δ subunit is in very close proximity to the γ subunit near the bottom of the F₁ molecule but does not penetrate deeply into the central core, (b) shows that within F₁ the δ subunit's N-terminus is exposed while its C-terminus is masked, (c) indicates that access to the δ subunit is shielded in part by the α, β, and γ subunits and changes during the catalytic cycle of F₁, and (d) implicates the δ subunit as important for the structural stability of the F₁ unit. These novel findings on a higher eukaryotic F₁-δ subunit are discussed in relationship to earlier studies on the related ε subunits from both chloroplasts and E. coli.