Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean.
A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis). RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD. Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains. RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM. Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid. Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80%. A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids. Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2.
Neelam
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A
School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom.
Marvier
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AC
Hall
J L
JL
Williams
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LE
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000494:pharmacology; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D002367:Ricinus communis / Q000201:enzymology / Q000235:genetics*; D002956:Citrulline / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D019143:Evolution, Molecular; D018628:Gene Dosage; D015870:Gene Expression; D015723:Gene Library; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D018076:DNA, Complementary; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011522:Protons; D011994:Recombinant Proteins; D002956:Citrulline