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J Exp Bot
2011 Aug 01;6212:4391-8. doi: 10.1093/jxb/err158.
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The aromatic/arginine selectivity filter of NIP aquaporins plays a critical role in substrate selectivity for silicon, boron, and arsenic.
Mitani-Ueno N
,
Yamaji N
,
Zhao FJ
,
Ma JF
.
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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.
Fig. 1. Alignment of the sequences of OsLsi1 and AtNIP5;1. The residues corresponding to NPA motifs and ar/R regions are boxed or shaded grey, respectively.
Fig. 2. Western blot analysis of native and mutated OsLsi1 and AtNIP5;1 proteins. Proteins were extracted from oocytes expressing OsLsi1, AtNIP5;1, and their mutants. A 15âμg aliquot of membrane fractions was resolved on a polyacrylamide gel and probed with anti-FLAG antibody. (A) Native OsLsi1 and its mutants: lane 1, water-injected negative control; lane 2, OsLsi1 original; lane 3, AIGR (G88A-S207I); lane 4, ASGR (G88A); lane 5, GIGR (S207I). (B) Native AtNIP5;1 and its mutants: lane 1, native AtNIP5;1; lane 2, GSGR (A117G-I236S); lane 3, GIGR (A117S); lane 4 ASGR (I236S); lane 5, NPA, NPA, GSGR (S139A-V250A, A117G-I236S).
Fig. 3. Influence of ar/R selectivity filter substitution of OsLsi1 on the transport activity for silicic acid (A), arsenite (B), boric acid (C), and water (D). Transport activity was determined for the water-injected negative control (H2O), oocytes expressing native OsLsi1 (GSGR original), or mutated proteins G88A (ASGR), S207I (GIGR), and G88A-S207I (AIGR). Data are means ±SD (n=3) for (A) and (B), (n=5â8) for (C) and (D). Different letters above the columns indicate statistically significant differences at P <0.05 by Tukey's test.
Fig. 4. Influence of ar/R selectivity filter substitution of AtNIP5;1 on the transport activity for silicic acid (A), arsenite (B), boric acid (C), and water (D). Transport activity was determined for the water-injected negative control (H2O), native AtNIP5;1 (AIGR original), A117G (GIGR), I236S (ASGR), and A117G-I236S (GSGR). Data are means ±SD (n=3) for (A) and (B), (n=5â6) for (C) and (D). Different letters above the columns indicate statistically significant differences at P <0.05 by Tukey's test.
Fig. 5. Influence of double substitution of NPA motifs and the ar/R selectivity filter of AtNIP5;1 on the transport activity for silicic acid (A), arsenite (B), boric acid (C), and water (D). Transport activity was determined for the water-injected negative control (H2O), native AtNIP5;1 (NPS, NPV, AIGR), or S139A-V250A and A117G-I236S (NPA, NPA, GSGR). Data are means ±SD (n=3) for (A) and (B), (n=5â6) for (C) and (D). Different letters above the columns indicate statistically significant differences at P <0.05 by Tukey's test.
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