title: Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.
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.
authors.affiliation: Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan, ROC.
authors.forename: Hsin Hung
authors.initials: HH
authors.lastname: Lin
authors.forename: Yih Jiuan
authors.initials: YJ
authors.lastname: Pan
authors.forename: Shen Hsing
authors.initials: SH
authors.lastname: Hsu
authors.forename: Ru Chuan
authors.initials: RC
authors.lastname: Van
authors.forename: Yi Yuong
authors.initials: YY
authors.lastname: Hsiao
authors.forename: Jiun Hsien
authors.initials: JH
authors.lastname: Chen
authors.forename: Rong Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D043564:Inorganic Pyrophosphatase / Q000235:genetics* / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology; D008670:Metals / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017494:Proton Pumps / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics*; D014617:Vacuoles / Q000201:enzymology* / Q000235:genetics
chemical_list: D008670:Metals; D010940:Plant Proteins; D017494:Proton Pumps; D043564:Inorganic Pyrophosphatase