Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan.
J Exp Bot. 2011 Aug;62(12):4391-8. doi: 10.1093/jxb/err158. Epub 2011 May 16.
Nodulin-26-like intrinsic proteins (NIPs) of the aquaporin family are involved in the transport of diverse solutes, but the mechanisms controlling the selectivity of transport substrates are poorly understood. The purpose of this study was to investigate how the aromatic/arginine (ar/R) selectivity filter influences the substrate selectivity of two NIP aquaporins; the silicic acid (Si) transporter OsLsi1 (OsNIP2;1) from rice and the boric acid (B) transporter AtNIP5;1 from Arabidopsis; both proteins are also permeable to arsenite. Native and site-directed mutagenized variants of the two genes were expressed in Xenopus oocytes and the transport activities for Si, B, arsenite, and water were assayed. Substitution of the amino acid at the ar/R second helix (H2) position of OsLsi1 did not affect the transport activities for Si, B, and arsenite, but that at the H5 position resulted in a total loss of Si and B transport activities and a partial loss of arsenite transport activity. Conversely, changes of the AtNIP5;1 ar/R selectivity filter and the NPA motifs to the OsLsi1 type did not result in a gain of Si transport activity. B transport activity was partially lost in the H5 mutant but unaffected in the H2 mutant of AtNIP5;1. In contrast, both the single and double mutations at the H2 and/or H5 positions of AtNIP5;1 did not affect arsenite transport activity. The results reveal that the residue at the H5 position of the ar/R filter of both OsLsi1 and AtNIP5;1 plays a key role in the permeability to Si and B, but there is a relatively low selectivity for arsenite.
水通道蛋白家族的类豆球蛋白-26 内在蛋白 (NIPs) 参与多种溶质的运输，但控制运输底物选择性的机制知之甚少。本研究旨在探讨芳香族/精氨酸（ar/R）选择性过滤器如何影响两种 NIP 水通道蛋白的底物选择性；来自水稻的硅（Si）转运蛋白 OsLsi1（OsNIP2;1）和来自拟南芥的硼酸（B）转运蛋白 AtNIP5;1；这两种蛋白也可渗透亚砷酸盐。在非洲爪蟾卵母细胞中表达了两种基因的天然和定点突变变体，并测定了 Si、B、亚砷酸盐和水的转运活性。OsLsi1 的 ar/R 第二螺旋（H2）位置的氨基酸取代不影响 Si、B 和亚砷酸盐的转运活性，但 H5 位置的取代导致 Si 和 B 转运活性完全丧失，亚砷酸盐转运活性部分丧失。相反，AtNIP5;1 ar/R 选择性过滤器和 NPA 基序的变化为 OsLsi1 类型不会导致 Si 转运活性增加。B 转运活性在 AtNIP5;1 的 H5 突变体中部分丧失，但在 H2 突变体中不受影响。相比之下，AtNIP5;1 的 H2 和/或 H5 位置的单个和双突变均不影响亚砷酸盐转运活性。结果表明，OsLsi1 和 AtNIP5;1 的 ar/R 过滤器 H5 位置的残基在 Si 和 B 的通透性中起关键作用，但对亚砷酸盐的选择性相对较低。
