ABSTRACT
This work describes the isolation of a full-length (VfAAP2) and three partial amino acid transporter genes (VfAAPa, VfAAPb, VfAAPc) from broad bean (Vicia faba L.). The function of VfAAP2 was tested by heterologous expression in a yeast mutant deficient in proline uptake. VfAAP2 mediates proton-dependent proline uptake with an apparent Km of about 1 mM. Analysis of substrate specificity by competition experiments showed that aromatic amino acids, neutral aliphatic acids and L-citrulline are the best competitors, whereas basic amino acids do not compete with proline. Northern analysis indicates that all VfAAPs exhibit different patterns of expression. VfAAP2 is most strongly expressed in the stem and at a lower level in sink leaves and pods. VfAAPa, VfAAPb and VfAAPc are most strongly expressed in the flowers, but their expression in the other organs varies.
Subject(s)
Carrier Proteins/genetics , Plant Proteins/genetics , Amino Acid Sequence , Amino Acid Transport Systems , Base Sequence , Biological Transport , Carrier Proteins/isolation & purification , Cloning, Molecular , Fabaceae , Gene Expression , Molecular Sequence Data , Plant Proteins/isolation & purification , Plants, Medicinal , Sequence Homology, Amino AcidABSTRACT
An 1.7-kb Arabidopsis thaliana (At) cDNA was isolated by complementation of a bap1 mutation affecting the transport of branched-chain amino acids (aa) in the yeast Saccharomyces cerevisiae. The determination of the nucleotide (nt) sequence revealed an open reading frame of 1383 nt which may encode a protein of 461 aa with a predicted molecular mass of 51,038 Da. The deduced aa sequence exhibited strong similarities with mammalian 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) sequences. Although former biochemical studies have suggested that acetoacetyl-coenzyme A thiolase (AACT) and HMGS activities were carried by a single protein in plants, complementation studies and measurements of enzymatic activities clearly showed that the At HMGS is devoid of AACT activity.