Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern, Switzerland.
J Biol Chem. 2010 Dec 17;285(51):39710-7. doi: 10.1074/jbc.M110.141457. Epub 2010 Oct 11.
The Arabidopsis di- and tripeptide transporters AtPTR1 and AtPTR5 were expressed in Xenopus laevis oocytes, and their selectivity and kinetic properties were determined by voltage clamping and by radioactive uptake. Dipeptide transport by AtPTR1 and AtPTR5 was found to be electrogenic and dependent on protons but not sodium. In the absence of dipeptides, both transporters showed proton-dependent leak currents that were inhibited by Phe-Ala (AtPTR5) and Phe-Ala, Trp-Ala, and Phe-Phe (AtPTR1). Phe-Ala was shown to reduce leak currents by binding to the substrate-binding site with a high apparent affinity. Inhibition of leak currents was only observed when the aromatic amino acids were present at the N-terminal position. AtPTR1 and AtPTR5 transport activity was voltage-dependent, and currents increased supralinearly with more negative membrane potentials and did not saturate. The voltage dependence of the apparent affinities differed between Ala-Ala, Ala-Lys, and Ala-Asp and was not conserved between the two transporters. The apparent affinity of AtPTR1 for these dipeptides was pH-dependent and decreased with decreasing proton concentration. In contrast to most proton-coupled transporters characterized so far, -I(max) increased at high pH, indicating that regulation of the transporter by pH overrides the importance of protons as co-substrate.
拟南芥二肽和三肽转运蛋白 AtPTR1 和 AtPTR5 在非洲爪蟾卵母细胞中表达，并通过电压钳和放射性摄取来确定其选择性和动力学特性。发现 AtPTR1 和 AtPTR5 对二肽的运输是电活性的，并且依赖于质子但不依赖于钠离子。在没有二肽的情况下，两种转运蛋白都显示出质子依赖性泄漏电流，该电流被 Phe-Ala（AtPTR5）和 Phe-Ala、Trp-Ala 和 Phe-Phe（AtPTR1）抑制。证明 Phe-Ala 通过与具有高表观亲和力的底物结合位点结合来减少泄漏电流。只有当芳香族氨基酸位于 N 末端位置时，才会观察到抑制泄漏电流。AtPTR1 和 AtPTR5 的转运活性是电压依赖性的，电流随着更负的膜电位呈超线性增加，并且不会饱和。Ala-Ala、Ala-Lys 和 Ala-Asp 的表观亲和力的电压依赖性在两个转运蛋白之间不同。AtPTR1 对这些二肽的表观亲和力随 pH 值的降低而降低。与迄今为止表征的大多数质子偶联转运蛋白不同，-I(max)在高 pH 值下增加，表明转运蛋白受 pH 值的调节超过质子作为共底物的重要性。
