CLONING OF A CDNA CODING FOR AN AMINO-ACID CARRIER FROM RICINUS-COMMUNIS (RCAAP1) BY FUNCTIONAL COMPLEMENTATION IN YEAST - KINETIC-ANALYSIS, INHIBITOR SENSITIVITY AND SUBSTRATE-SPECIFICITY

A cDNA for the amino acid permease gene RcAAP1 has been isolated from Ricinus communis by yeast complementation and subjected to a detailed kinetic analysis. RcAAP1 cDNA is 1.5 kb with an open reading frame tha t codes for a protein with 486 amino acids and a calculated molecular mass of 53.1 kDa. RcAAP1-mediated histidine uptake was pH dependent wi th highest transport rates at acidic pH; it was sensitive to protonoph ores and uncouplers and the K-m for histidine uptake was 96 mu M. The substrate specificity was investigated by measuring the levels of inhi bition of histidine uptake by a range of amino acids. The basic amino acids (histidine, lysine and arginine) showed strongest inhibition of uptake whereas acidic amino acids competed less effectively. Alanine w as the most efficient competitor of the neutral amino acids. Glutamine , serine, asparagine, methionine and cysteine showed moderate inhibiti on whereas threonine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan showed only low levels of inhibition. Glycine, proline and citrulline caused slight stimulation. More detailed competition kinet ics indicated that both lysine and arginine showed simple competitive inhibition of histidine uptake. When direct uptake measurements were c arried out, both lysine and arginine were found to be effective substr ates for RcAAP1. (C) 1998 Elsevier Science B.V. All rights reserved.