Möhlmann T, Tjaden J, Schwöppe C, Winkler H H, Kampfenkel K, Neuhaus H E
Pflanzenphysiologie, Universität Osnabrück, Germany.
Eur J Biochem. 1998 Mar 15;252(3):353-9. doi: 10.1046/j.1432-1327.1998.2520353.x.
Recently, we sequenced a cDNA clone from Arabidopsis thaliana L. encoding an ATP/ADP transporter protein (AATP1) located in the plastid envelope membrane. The deduced amino acid sequence of AATP1 exhibits a high degree of similarity (> 66%) to the ATP/ADP transporter from the obligate intracellular gram-negative bacterium Rickettsia prowazekii. Here we report a second plastidic ATP/ADP carrier from A. thaliana (AATP2). As deduced from the amino acid sequence, AATP2 exhibits 77.6% identity to AATP1 and 36% to the rickettsial protein. Hydropathy analysis indicates that all three translocators are highly hydrophobic membrane proteins, which exhibit marked similarities and differences. The AATP1 translocator lacks the sixth transmembrane domain that is present in AATP2 and the bacterial transporter in R. prowazekii. In contrast to AATP1 and the bacterial transport protein, only AATP2 exhibits a truncated C-terminal end. To compare the general biochemical properties of AATP2 with the known transport properties of AATP1 we cloned the entire AATP2 cDNA into plasmid pJT118, leading to the presence of an additional N-terminal histidine tag of 10 amino acids. For heterologous expression of His10-AATP2 we chose the Escherichia coli strain C43, which was reported recently to allow overproduction of eucaryotic membrane transport proteins. After transformation and subsequent induction by isopropylthio-2-D-galactopyranoside intact E. coli cells harbouring plasmid pJT118 showed import of radioactively labelled ATP and ADP. As deduced from a Lineweaver-Burk analysis His10-AATP2 exhibited apparent Km values for ATP and ADP of 22 microM and 20 microM, respectively. Import of ADP into His10-AATP2-expressing E. coli cells occurred at a rate of 24 nmol x mg protein(-1) x h(-1), which was about threefold faster than import of ATP. These biochemical characteristics are similar to transport properties of the heterologously expressed His10-AATP1 protein.
最近，我们对来自拟南芥的一个cDNA克隆进行了测序，该克隆编码一种位于质体包膜膜上的ATP/ADP转运蛋白（AATP1）。AATP1推导的氨基酸序列与专性细胞内革兰氏阴性细菌普氏立克次体的ATP/ADP转运蛋白具有高度相似性（>66%）。在此，我们报道了拟南芥的第二种质体ATP/ADP载体（AATP2）。从氨基酸序列推断，AATP2与AATP1的同一性为77.6%，与立克次体蛋白的同一性为36%。亲水性分析表明，所有这三种转运体都是高度疏水的膜蛋白，它们表现出明显的相似性和差异。AATP1转运体缺少AATP2和普氏立克次体中的细菌转运体所具有的第六个跨膜结构域。与AATP1和细菌转运蛋白不同，只有AATP2的C末端是截短的。为了将AATP2的一般生化特性与已知的AATP1转运特性进行比较，我们将整个AATP2 cDNA克隆到质粒pJT118中，导致出现了一个额外的10个氨基酸的N末端组氨酸标签。为了实现His10 - AATP2的异源表达，我们选择了大肠杆菌C43菌株，最近有报道称该菌株能够过量表达真核膜转运蛋白。在转化并随后用异丙基硫代 - 2 - D - 半乳糖吡喃糖苷诱导后，携带质粒pJT118的完整大肠杆菌细胞显示出对放射性标记的ATP和ADP的摄取。从Lineweaver - Burk分析推断，His10 - AATP2对ATP和ADP的表观Km值分别为22 microM和20 microM。ADP进入表达His10 - AATP2的大肠杆菌细胞的速率为24 nmol·mg蛋白⁻¹·h⁻¹，这比ATP的摄取速率快约三倍。这些生化特性与异源表达的His10 - AATP1蛋白的转运特性相似。
