Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L
Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L
复制标题
DOI：
10.1046/j.1365-313x.1997.11010073.x
复制
发表时间：
1997-01-01
期刊：
影响因子：
7.2
通讯作者：
Kampfenkel, K
中科院分区：
文献类型：
--
作者：
Neuhaus, HE;Thom, E;Kampfenkel, K
最近，我们对拟南芥（Arabidopsis thaliana L.）编码一种新的推定的ATP/ADP转运蛋白（AATP 1）。在这里，我们证明了放射性标记的AATP 1前体蛋白，在体外合成，有针对性的包膜膜分离菠菜叶绿体。针对AATP 1的合成肽产生的抗体在叶绿体内被膜制剂中识别约62 kDa的单一多肽。编码AATP 1蛋白的cDNA在酿酒酵母和大肠杆菌中功能性表达。在这两种表达系统中，将提取的蛋白质重组到脂蛋白体中后，观察到ATP转运速率增加。据我们所知，这是第一个关于植物内在膜蛋白在大肠杆菌中功能表达的报道。杆菌为了产生高速率的ATP转运，蛋白脂质体必须预先装载ADP，这表明转运的逆交换模式。羧基糖苷没有实质性干扰ATP转运到含有质体ATP/ADP转运蛋白的蛋白脂质体。测定ATP的表观K-M为28 μ M，这与报道的分离质体的值相似。这里提出的数据强烈支持的结论，AATP 1代表一种新的真核腺苷酸载体，它是迄今未知的质体ATP/ADP转运相同。
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 K-M for ATP of 28 mu M 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.