Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana.
The energy-dependent transport of solutes across the vacuolar membrane (tonoplast) of plant cells is driven by two H+ pumps: a vacuolar ("V-type") H(+)-ATPase (EC 3.6.1.3) and a H(+)-translocating (pyrophosphate-energized) inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1). The H(+)-PPase, like the V-type H(+)-ATPase, is abundant and ubiquitous in the vacuolar membranes of plant cells, and both enzymes make a substantial contribution to the transtonoplast H(+)-electrochemical potential difference. Here, we report the cloning and sequence of cDNAs encoding the tonoplast H(+)-PPase of Arabidopsis thaliana. The protein predicted from the nucleotide sequence of the cDNAs is constituted of 770 amino acids and has a molecular weight of 80,800. It is a highly hydrophobic integral membrane protein, and the structure derived from hydrophilicity plots contains at least 13 transmembrane spans. Since the tonoplast H(+)-PPase appears to be constituted of one polypeptide species and genomic Southern analyses indicate that the gene encoding the Mr 80,800 polypeptide is present in only a single copy in the genome of Arabidopsis, it is suggested that the H(+)-PPase has been cloned in its entirety. The lack of sequence identities between the tonoplast H(+)-PPase and any other characterized H+ pump or PPi-dependent enzyme implies a different evolutionary origin for this translocase.
Sarafian
V
V
Department of Biology, University of Pennsylvania, Philadelphia 19104.
Kim
Y
Y
Poole
R J
RJ
Rea
P A
PA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D043564:Inorganic Pyrophosphatase; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics*; D011755:Pyrophosphatases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012995:Solubility; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure
D011756:Diphosphates; D008565:Membrane Proteins; D012333:RNA, Messenger; D004247:DNA; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase