Mathieu Christoph, Macêdo Juan P, Hürlimann Daniel, Wirdnam Corina, Haindrich Alexander C, Suter Grotemeyer Marianne, González-Salgado Amaia, Schmidt Remo S, Inbar Ehud, Mäser Pascal, Bütikofer Peter, Zilberstein Dan, Rentsch Doris
Institute of Plant Sciences, University of Bern, Bern, Switzerland.
Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.
PLoS One. 2017 Jan 3;12(1):e0168775. doi: 10.1371/journal.pone.0168775. eCollection 2017.
For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.
对于布氏锥虫而言，精氨酸和赖氨酸是必需氨基酸，因此必须从宿主中摄取。在酿酒酵母突变体中的异源表达在布氏锥虫AAAP（氨基酸/生长素通透酶）家族成员中鉴定出阳离子氨基酸转运蛋白。TbAAT5-3表现出对精氨酸的高亲和力摄取（Km为3.6±0.4μM）以及对L-精氨酸的高选择性。L-精氨酸的转运可被10倍过量的L-精氨酸、高瓜氨酸、刀豆氨酸或精氨酸-β-萘酰胺所降低，而赖氨酸仅在100倍过量时具有抑制作用，组氨酸或鸟氨酸不会显著降低精氨酸的摄取速率。TbAAT16-1是一种高亲和力（Km为4.3±0.5μM）且高度选择性的L-赖氨酸转运蛋白，在所测试的化合物中，只有L-赖氨酸和硫代赖氨酸竞争L-赖氨酸的摄取。TbAAT5-3和TbAAT16-1在布氏锥虫的前循环型和血流型中均有表达，带有cMyc标签的蛋白表明其定位于质膜。RNA干扰介导的血流型锥虫中TbAAT5和TbAAT16的下调导致生长停滞，这表明TbAAT5介导的精氨酸转运和TbAAT16介导的赖氨酸转运对布氏锥虫至关重要。分别补充过量的精氨酸或赖氨酸可部分挽救诱导RNA干扰品系的生长，而同时添加这两种氨基酸的效果较差。单RNA干扰和双RNA干扰品系表明布氏锥虫中存在额外的低亲和力精氨酸和赖氨酸摄取系统。
