Article

Acta Biochim Biophys Sin 2005,37:453-462

doi:10.1111/j.1745-7270.2005.00064.x

Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase e Subunit

Hui DONG, Zhang-Lin NI, and Jia-Mian WEI*

Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China

Abstract The conserved residue Gly47 of the chloroplast ATP synthase e subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants eG47L, eG47R, eG47A and eG47E, respectively. All the e variants showed lower inhibitory effects on the soluble CF1(–e) Ca2+-ATPase compared with wild-type e. In reduced conditions, eG47E and eG47R had a lower inhibitory effect on the oxidized CF1(–e) Ca2+-ATPase compared with wild-type e. In contrast, eG47L and eG47A increased the Ca2+-ATPase activity of soluble oxidized CF1(–e). The replacement of Gly47 significantly impaired the interaction between the subunit e and g in an in vitro binding assay. Further study showed that all e 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 e. These results indicate that the conserved Gly47 residue of the e subunit is very important for maintaining the structure and function of the e subunit and may affect the interaction between the e subunit, b subunit of CF1 and subunit III of CFo, thereby regulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit III of CFo.

Key words chloroplast; ATP synthase; e subunit; GST pull-down assay; site-directed mutagenesis

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