Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase |
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Authors: | Lin Hsin Hung Pan Yih Jiuan Hsu Shen Hsing Van Ru Chuan Hsiao Yi Yuong Chen Jiun Hsien Pan Rong Long |
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Institution: | Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan, ROC. |
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Abstract: | Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner. |
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Keywords: | Proton translocation Proton translocase Tonoplast Vacuole Vacuolar H+-pyrophosphatase Inorganic pyrophosphate Heterologous expression C-terminal truncation Deletion mutagenesis K+-binding |
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