Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.
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.
du Jardin
P
P
Department of Plant Biology, Faculty of Agricultural Sciences of Gembloux, Belgium.
Rojas-Beltran
J
J
Gebhardt
C
C
Brasseur
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D017343:Genes, Plant; D005816:Genetic Complementation Test; D043564:Inorganic Pyrophosphatase; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D005810:Multigene Family; D012150:Polymorphism, Restriction Fragment Length; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium