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.
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