Open AccessSubstitutions of the Conserved Gly47 Affect the CF 1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ɛ Subunit
Author(s) -
DONG Hui,
NI ZhangLin,
WEI JiaMian
Publication year - 2005
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2005.00070.x
Subject(s) - atp synthase gamma subunit , atp synthase , protein subunit , gamma subunit , chloroplast , atp hydrolysis , atpase , mutant , thylakoid , wild type , chemistry , biochemistry , biology , enzyme , gene
The conserved residue Gly47 of the chloroplast ATP synthase ɛ subunit was substituted with Leu, Arg, Ala and Glu by site‐directed mutagenesis. This process generated the mutants ɛG47L, ɛG47R, ɛG47A and ɛG47E, respectively. All the ɛ variants showed lower inhibitory effects on the soluble CF 1 (–ɛ) Ca 2+ ‐ATPase compared with wild‐type ɛ. In reduced conditions, ɛG47E and ɛG47R had a lower inhibitory effect on the oxidized CF1(–ɛ) Ca 2+ ‐ATPase compared with wild‐type ɛ. In contrast, ɛG47L and ɛG47A increased the Ca 2+ ‐ATPase activity of soluble oxidized CF 1 (–ɛ). The replacement of Gly47 significantly impaired the interaction between the subunit ɛ and γ in an in vitro binding assay. Further study showed that all ɛ variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild‐type ɛ. These results indicate that the conserved Gly47 residue of the ɛ subunit is very important for maintaining the structure and function of the ɛ subunit and may affect the interaction between the ɛ subunit, β subunit of CF 1 and subunit III of CF o , thereby regulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit III of CF o . Edited by Wen‐Hua ZHANG