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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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