PLANT PHYSIOLOGY , Vol 109, Issue 3 853-860, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Molecular Cloning and Characterization of a Soluble Inorganic Pyrophosphatase in Potato
P. du Jardin, J. Rojas-Beltran, C. Gebhardt and R. Brasseur
Department of Plant Biology (P.d.J., J.R.-B.) and Center of Numerical Molecular Biophysics (R.B.), Faculty of Agricultural Sciences of Gembloux, B-5030 Gembloux, Belgium
A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of
potato (Solanum tuberosum L.) was isolated by screening a developing tuber
library with a heterologous probe. The central domain of the encoded
polypeptide is nearly identical at the sequence level with its Arabidopsis
homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348).
Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli
soluble pyrophosphatases indicated a remarkably conserved organization of
the hydrophobic protein domains. The enzymatic function of the potato
protein could be deduced from the presence of amino acid residues highly
conserved in soluble pyrophosphatases and was confirmed by its capacity to
complement a thermosensitive pyrophosphatase mutation in E. coli. The
potato polypeptide was purified from complemented bacterial cells and its
pyrophosphatase activity was shown to be strictly dependent on Mg2+ and
strongly inhibited by Ca2+. The subcellular location of the potato
pyrophosphatase is unknown. Structure analysis of the N-terminal protein
domain failed to recognize typical transit peptides and the calculated
molecular mass of the polypeptide (24 kD) is significantly inferior to the
values reported for the plastidic (alkaline) or mitochondrial
pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by
restriction fragment length polymorphism analysis in the potato genome
using the full-length cDNA as probe.
