Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.

Authors

  • H.E. Neuhaus,

    Corresponding author
    1. Pflanzenphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastraße 11, D-49069 Osnabrück, Germany
    Search for more papers by this author
  • E. Thom,

    1. Institut für Biochemie und Molekulare Physiologie, Universität Potsdam, Maulbeerallee 2a, D-14469 Potsdam, Germany
    Search for more papers by this author
  • T. Möhlmann,

    1. Pflanzenphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastraße 11, D-49069 Osnabrück, Germany
    Search for more papers by this author
  • M. Steup,

    1. Institut für Biochemie und Molekulare Physiologie, Universität Potsdam, Maulbeerallee 2a, D-14469 Potsdam, Germany
    Search for more papers by this author
  • K. Kampfenkel

    1. Institut für Biochemie und Molekulare Physiologie, Universität Potsdam, Maulbeerallee 2a, D-14469 Potsdam, Germany
    Search for more papers by this author
    • Present address: Novo Nordisk AS, Novo Allé, DK 2880 Bagsvaerd, Denmark.


*For correspondence (fax +49 541 9692870; e-mail: Neuhaus@sfbbio 1.biologie.uni-osnabrueck.de).

Abstract

Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L. encoding a novel putative ATP/ADP translocator (AATP1). Here, we demonstrate that the radioactively labeled AATP1 precursor protein, synthesized in vitro, is targeted to envelope membranes of isolated spinach chloroplasts. Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of about 62 kDa in chloroplast inner envelope preparations. The cDNA coding for the AATP1 protein was functionally expressed in Saccharomyces cerevisiae and Escherichia coli. In both expression systems, increased rates of ATP transport were observed after reconstitution of the extracted protein into proteoliposomes. To our knowledge, this is the first report on the functional expression of an intrinsic plant membrane protein in E. coli. To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indicating a counter-exchange mode of transport. Carboxyatractyloside did not substantially interfere with ATP transport into proteoliposomes containing the plastidic ATP/ADP translocator. An apparent KM for ATP of 28 µM was determined which is similar to values reported for isolated plastids. The data presented here strongly support the conclusion that AATP1 represents a novel eukaryotic adenylate carrier and that it is identical with the so far unknown plastidic ATP/ADP translocator.

Ancillary