Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.
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.
Lin
Hsin Hung
HH
Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan, ROC.
Pan
Yih Jiuan
YJ
Hsu
Shen Hsing
SH
Van
Ru Chuan
RC
Hsiao
Yi Yuong
YY
Chen
Jiun Hsien
JH
Pan
Rong Long
RL
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
D008670:Metals; D010940:Plant Proteins; D017494:Proton Pumps; D043564:Inorganic Pyrophosphatase