Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.
The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H(+)-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H(+)-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H+ translocation is not known. Here we address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H+ translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H+ translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP.
Department of Biology, University of Pennsylvania, Philadelphia 19104.
E J
EJ
Kim
R G
RG
Zhen
P A
PA
Rea
D000595:Amino Acid Sequence; D000906:Antibodies; D017360:Arabidopsis / Q000201:enzymology*; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology; D005459:Fluorides / Q000494:pharmacology; D005822:Genetic Vectors; D043564:Inorganic Pyrophosphatase; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010946:Plants, Medicinal; D011522:Protons; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D000906:Antibodies; D046911:Macromolecular Substances; D010455:Peptides; D011522:Protons; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D005459:Fluorides