Effects of sequential deletions of residues from the N- or C-terminus on the functions of epsilon subunit of the chloroplast ATP synthase

Ten truncated mutants of chloroplast ATP synthase c subunit from spinach (S pinacia oleracea), which had sequentially lost 1-5 amino acid residues from the N-terminus and 6-10 residues from the C-terminus, were generated by PC R. These mutants were overexpressed in Escherichia coli, reconstituted with soluble and membrane-bound CF1, and the ATPase activity and proton conduct ance of thylakoid membrane were examined. Deletions of as few as 3 amino ac id residues from the N-terminus or 6 residues from the C-terminus of epsilo n subunit significantly affected their ATPase-inhibitory activity in soluti on. Deletion of 5 residues from the N-terminus abolished its abilities to i nhibit ATPase activity and to restore proton impermeability. Considering th e consequence of interaction of epsilon and gamma subunit in the enzyme fun ctions, the special interactions between the epsilon variants and the gamma subunit were detected in the yeast two-hybrid system and in vitro binding assay. In addition, the structures of these mutants were modeled through th e SWISS-MODEL Protein Modeling Server. These results suggested that in chlo roplast ATP synthase, both the N-terminus and C-terminus of the epsilon sub unit show importance in regulation of the ATPase activity. Furthermore, the N-terminus of the epsilon subunit is more important for its interaction wi th gamma and some CFo subunits, and crucial for the blocking of proton leak age. Compared with the epsilon subunit from E. coli [Jounouchi, M., Takeyam a, M., Noumi:ii, T., Moriyama, Y., Maeda, M., and Futai, M. (1992) Arch. Bi ochem. Biophys. 292, 87-94; Kuki, M., Noumi, T., Maeda, M., Amemura, A., an d Futai, M. (1988) J. Biol. Chem. 263, 4335-4340], the chloroplast 6 subuni t is more sensitive to N-terminal or C-terminal truncations.