N-terminal deletion of the γ subunit affects the stabilization and activity of chloroplast ATP synthase

Abstract

Five truncation mutants of chloroplast ATP synthase γ subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinant γ genes were overexpressed in Escherichia coli and assembled with αβ subunits into a native complex. The wild-type (WT) αβγ assembly i.e. αβγWT exhibited high Mg2+-dependent and Ca 2+-dependent ATP hydrolytic activity. Deletions of eight residues of the γ subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the αβWT assembly. Furthermore, only ≈6% of ATP hydrolytic activity was retained with the sequential deletions of γ subunit up to 20 residues compared with the activity of the αβWT assembly. The inhibitory effect of the ε subunit on ATP hydrolysis of these αβγ assemblies varied to a large extent. These observations indicate that the N-terminus of the γ subunit is very important, together with other regions of the γ subunit, in stabilization of the enzyme complex or during cooperative catalysis. In addition, the in vitro binding assay showed that the γ subunit N-terminus is not a crucial region in binding of the ε subunit. © 2005 FEBS.