Substitutions of the conserved Gly47 affect the CF1 inhibitor and proton gate functions of the chloroplast ATP synthase epsilon subunit.
The conserved residue Gly47 of the chloroplast ATP synthase beta subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants epsilon G47L, epsilon G47R, epsilon G47A and epsilon G47E, respectively. All the beta variants showed lower inhibitory effects on the soluble CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In reduced conditions, epsilon G47E and epsilon G47R had a lower inhibitory effect on the oxidized CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In contrast, epsilon G47L and epsilon G47A increased the Ca2+-ATPase activity of soluble oxidized CF1(-epsilon). The replacement of Gly47 significantly impaired the interaction between the subunit epsilon and gamma in an in vitro binding assay? Further study showed that all epsilon 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 epsilon. These results indicate that the conserved Gly47 residue of the epsilon subunit is very important for maintaining the structure and function of the epsilon subunit and may affect the interaction between the epsilon subunit, beta 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.
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Hui
H
Dong
Zhang-Lin
ZL
Ni
Jia-Mian
JM
Wei
D025222:Chloroplast Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D002736:Chloroplasts / Q000201:enzymology; D005998:Glycine / Q000737:chemistry*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010785:Photophosphorylation; D011506:Proteins / Q000235:genetics*; D011522:Protons; D018724:Spinacia oleracea / Q000201:enzymology / Q000648:ultrastructure; D020524:Thylakoids / Q000378:metabolism; D000097795:ATPase Inhibitory Protein
D025222:Chloroplast Proton-Translocating ATPases; D005998:Glycine; D011506:Proteins; D011522:Protons; D000097795:ATPase Inhibitory Protein