| Abstract
| - Ten truncated mutants of chloroplast ATP synthase ε subunit from spinach (Spinacia oleracea),which had sequentially lost 1−5 amino acid residues from the N-terminus and 6−10 residues from theC-terminus, were generated by PCR. These mutants were overexpressed in Escherichia coli, reconstitutedwith soluble and membrane-bound CF1, and the ATPase activity and proton conductance of thylakoidmembrane were examined. Deletions of as few as 3 amino acid residues from the N-terminus or 6 residuesfrom the C-terminus of ε subunit significently affected their ATPase-inhibitory activity in solution. Deletionof 5 residues from the N-terminus abolished its abilities to inhibit ATPase activity and to restore protonimpermeability. Considering the consequence of interaction of ε and γ subunit in the enzyme functions,the special interactions between the ε variants and the γ subunit were detected in the yeast two-hybridsystem and in vitro binding assay. In addition, the structures of these mutants were modeled through theSWISS-MODEL Protein Modeling Server. These results suggested that in chloroplast ATP synthase,both the N-terminus and C-terminus of the ε subunit show importance in regulation of the ATPase activity.Furthermore, the N-terminus of the ε subunit is more important for its interaction with γ and some CFosubunits, and crucial for the blocking of proton leakage. Compared with the ε subunit from E. coli[Jounouchi, M., Takeyama, M., Noumi, T., Moriyama, Y., Maeda, M., and Futai, M. (1992) Arch. Biochem.Biophys.292, 87−94; Kuki, M., Noumi, T., Maeda, M., Amemura, A., and Futai, M. (1988) J. Biol.Chem.263, 4335−4340], the chloroplast ε subunit is more sensitive to N-terminal or C-terminal truncations.
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