Effects of site-directed mutation on the function of the chloroplast ATP synthase epsilon subunit.
The previous work in our lab showed that the spinach chloroplast ATP synthase epsilon mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast epsilon subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast epsilon subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants epsilonL3I, epsilonL3F, epsilonL3T, epsilonL3R, epsilonL3E and epsilonL3P, respectively. These epsilon variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type epsilon subunit (epsilonWT). The abilities of mutants epsilonL3I and epsilonL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of epsilonWT, but the abilities of the other epsilon variants were lower than that of epsilonWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast epsilon subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.
Zeng
Xiaomei
X
Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences.
Ni
Zhanglin
Z
Shi
Xiaobing
X
Wei
Jiamian
J
Shen
Yungang
Y
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D025222:Chloroplast Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D030781:Organisms, Genetically Modified; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology; D020524:Thylakoids / Q000378:metabolism
D021122:Protein Subunits; D000255:Adenosine Triphosphate; D025222:Chloroplast Proton-Translocating ATPases