CHARACTERIZATION OF A NOVEL EUKARYOTIC ATP ADP TRANSLOCATOR LOCATED IN THE PLASTID ENVELOPE OF ARABIDOPSIS-THALIANA L/

Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L. encoding a novel putative ATP/ADP translocator (AATP1). Here, we demon strate that the radioactively labeled AATP1 precursor protein, synthes ized in vitro, is targeted to envelope membranes of isolated spinach c hloroplasts. Antibodies raised against a synthetic peptide of AATP1 re cognized a single polypeptide of about 62 kDa in chloroplast inner env elope preparations. The cDNA coding for the AATP1 protein was function ally expressed in Saccharomyces cerevisiae and Escherichia coil In bot h expression systems, increased rates of ATP transport were observed a fter reconstitution of the extracted protein into proteoliposomes. To our knowledge, this is the first report on the functional expression o f an intrinsic plant membrane protein in E. coil. To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indica ting a counter-exchange mode of transport. Carboxyatractyloside did no t substantially interfere with ATP transport into proteoliposomes cont aining the plastidic ATP/ADP translocator. An apparent K-M for ATP of 28 mu M was determined which is similar to values reported for isolate d plastids. The data presented here strongly support the conclusion th at AATP1 represents a novel eukaryotic adenylate carrier and that it i s identical with the so far unknown plastidic ATP/ADP translocator.