Regina Teresa Maria Rosaria, Galluccio Michele, Scalise Mariafrancesca, Pochini Lorena, Indiveri Cesare
Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036, Arcavacata di Rende, Cosenza, Italy.
Plant Mol Biol. 2017 Aug;94(6):657-667. doi: 10.1007/s11103-017-0632-6. Epub 2017 Jul 10.
The vacuolar SlCAT2 was cloned, over-produced in E. coli and reconstituted in proteoliposomes. Arg, Ornithine and Lys were identified as substrates. Unexpectedly, also the organic cations Tetraethylammonium and Acetylcholine were transported indicating involvement of SlCAT2 in signaling. In land plants several transporters are involved in ion and metabolite flux across membranes of cells or intracellular organelles. The vacuolar amino acid transporter CAT2 from Solanum lycopersicum was investigated in this work. SlCAT2 was cloned from tomato flower cDNA, over-produced in Escherichia coli and purified by Nichel-chelating chromatography. For functional studies, the transporter was reconstituted in proteoliposomes. Competence of SlCAT2 for Arg transport was demonstrated measuring uptake of [H]Arg in proteoliposomes which was trans-stimulated by internal Arg or ornithine. Uptake of [H]Ornithine and [H]Lys was also detected at lower efficiency with respect to [H]Arg. Transport was activated by the presence of intraliposomal ATP suggesting regulation by the nucleotide. The prototype for organic cations tetraethylammonium (TEA) was also transported by SlCAT2. However, scarce reciprocal inhibition between TEA and Arg was found, while the biguanide metformin was able to strongly inhibit uptake of both substrates. These findings suggest that amino acids and organic cations may interact with the transporter through different functional groups some of which are common for the two types of substrates. Interestingly, reconstituted SlCAT2 showed competence for acetylcholine transport, which was also inhibited by metformin. Kinetics of Arg and Ach transport were performed from which Km values of 0.29 and 0.79 mM were derived, respectively.
液泡型SlCAT2被克隆，在大肠杆菌中过量表达并重组到蛋白脂质体中。精氨酸、鸟氨酸和赖氨酸被鉴定为底物。出乎意料的是，有机阳离子四乙铵和乙酰胆碱也能被转运，这表明SlCAT2参与了信号传导。在陆地植物中，几种转运蛋白参与离子和代谢物跨细胞膜或细胞内细胞器膜的流动。本研究对来自番茄的液泡氨基酸转运蛋白CAT2进行了研究。SlCAT2从番茄花cDNA中克隆，在大肠杆菌中过量表达，并通过镍螯合层析纯化。为了进行功能研究，将该转运蛋白重组到蛋白脂质体中。通过测量蛋白脂质体中[H]精氨酸的摄取来证明SlCAT2对精氨酸转运的能力，其摄取受到内部精氨酸或鸟氨酸的反刺激。相对于[H]精氨酸，[H]鸟氨酸和[H]赖氨酸的摄取效率也较低。脂质体内ATP的存在激活了转运，表明受核苷酸调节。有机阳离子四乙铵（TEA）的原型也能被SlCAT2转运。然而，在TEA和精氨酸之间发现了微弱的相互抑制作用，而双胍类药物二甲双胍能够强烈抑制两种底物的摄取。这些发现表明，氨基酸和有机阳离子可能通过不同的官能团与转运蛋白相互作用，其中一些官能团是这两种底物共有的。有趣的是，重组后的SlCAT2显示出对乙酰胆碱转运的能力，这也受到二甲双胍的抑制。进行了精氨酸和乙酰胆碱转运的动力学研究，分别得出Km值为0.29和0.79 mM。
