Tjaden J, Schwöppe C, Möhlmann T, Quick P W, Neuhaus H E
Pflanzenphysiologie, Universität Osnabrück, Barbarastr. 11, D-49069 Osnabrück, Germany.
J Biol Chem. 1998 Apr 17;273(16):9630-6. doi: 10.1074/jbc.273.16.9630.
Recently, a second type of eucaryotic adenine nucleotide transporter located in the inner envelope membrane of higher plants has been identified at the molecular level (Neuhaus, H. E., Thom, E., Möhlmann, T., Steup, M., and Kampfenkel, K. (1997) Plant J. 11, 73-82). Here we have analyzed the biochemical properties of this ATP/ADP transporter from Arabidopsis thaliana (AATP1, At). This analysis was carried out by expressing a cDNA encoding this carrier as a histidine-tagged chimeric protein heterologously in Escherichia coli. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-induced E. coli cells were able to import radioactively labeled [alpha-32P]ATP. Uninduced E. coli cells did not import [alpha-32P]ATP. Further control experiments revealed that IPTG induction did not promote import of other phosphorylated or unphosphorylated metabolites into the bacterial cell indicating the specificity of [alpha-32P]ATP transport. [alpha-32P]ATP uptake into induced E. coli cells was linear with time for several minutes allowing for determination of kinetic constants. The apparent Km for ATP was 17 microM which is close to values reported on the authentic protein in isolated plastids. ADP was a strong competitive inhibitor of -alpha-32P-ATP uptake (Ki ADP 3.6 microM). Other metabolites like AMP, ADP glucose, UTP, UDP, NAD, and NADP did not influence [alpha-32P]ATP uptake. IPTG-induced E. coli cells preloaded with [alpha-32P]ATP exported radioactively labeled adenylates after exogenous addition of unlabeled ATP or ADP indicating a counter exchange mechanism of transport. The biochemical properties of the heterologously expressed AATP1 gene product demonstrated that the protein is functionally integrated in the cytoplasmic membrane of E. coli. This is the first report of the functional expression of a plant membrane protein in E. coli leading to new transport properties across the cytoplasmic membrane. The functional integration of a plant membrane protein in the cytoplasmic membrane of E. coli offers new possibilities for future studies of the structural and mechanistic properties of this transporter. Since IPTG induction allowed synthesis of a 67-kDa protein in E. coli, which was subsequently specifically enriched by metal-chelate chromatography, this procaryotic heterologous expression system might provide a suitable system for overproduction of membrane proteins of eucaryotic origin in the near future.
最近，在分子水平上鉴定出了高等植物内膜中存在的第二种真核腺嘌呤核苷酸转运蛋白（Neuhaus, H. E., Thom, E., Möhlmann, T., Steup, M., and Kampfenkel, K. (1997) Plant J. 11, 73 - 82）。在此，我们分析了拟南芥中这种ATP/ADP转运蛋白（AATP1, At）的生化特性。该分析通过在大肠杆菌中异源表达编码这种载体的cDNA作为组氨酸标签嵌合蛋白来进行。异丙基 - 1 - 硫代 - β - D - 半乳糖苷（IPTG）诱导的大肠杆菌细胞能够摄取放射性标记的[α - 32P]ATP。未诱导的大肠杆菌细胞不摄取[α - 32P]ATP。进一步的对照实验表明，IPTG诱导并未促进其他磷酸化或未磷酸化代谢物进入细菌细胞，这表明了[α - 32P]ATP转运的特异性。诱导的大肠杆菌细胞摄取[α - 32P]ATP在几分钟内与时间呈线性关系，从而可以测定动力学常数。ATP的表观Km为17μM，这与分离的质体中真实蛋白报道的值相近。ADP是α - 32P - ATP摄取的强竞争性抑制剂（Ki ADP 3.6μM）。其他代谢物如AMP、ADP葡萄糖、UTP、UDP、NAD和NADP不影响[α - 32P]ATP摄取。预先加载了[α - 32P]ATP的IPTG诱导的大肠杆菌细胞在加入未标记的ATP或ADP后会输出放射性标记的腺苷酸，这表明存在反向交换转运机制。异源表达的AATP1基因产物的生化特性表明该蛋白在大肠杆菌细胞质膜中功能整合。这是关于植物膜蛋白在大肠杆菌中功能表达并导致跨细胞质膜产生新转运特性的首次报道。植物膜蛋白在大肠杆菌细胞质膜中的功能整合为该转运蛋白的结构和机制特性的未来研究提供了新的可能性。由于IPTG诱导可使大肠杆菌中合成一种67 kDa的蛋白，随后通过金属螯合层析进行特异性富集，这种原核异源表达系统可能在不久的将来为过量生产真核来源的膜蛋白提供一个合适的系统。
