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N‐terminal deletion of the γ subunit affects the stabilization and activity of chloroplast ATP synthase
Author(s) -
Ni ZhangLin,
Dong Hui,
Wei JiaMian
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04570.x
Subject(s) - atp synthase gamma subunit , protein subunit , gamma subunit , atp synthase , chloroplast , spinacia , atp hydrolysis , biochemistry , biology , c terminus , specificity factor , mutagenesis , mutant , microbiology and biotechnology , gamma aminobutyric acid receptor subunit alpha 1 , enzyme , escherichia coli , amino acid , gene , rna polymerase , g alpha subunit , atpase
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 Mg 2+ ‐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 οf 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.