Molecular analysis of ion transporters in plant and cyanobacteria involved in response to high osmolality

1.我们已经确定了拟南芥Na转运蛋白AtHKT1在植物细胞中的定位。证实了抗AtHKT1抗体的特异性。免疫电子显微镜显示，AtHKT1定位于叶片木质部薄壁细胞的质膜上。ATHKT1中断等位基因使木质部汁液中的钠含量显著增加，反之，韧皮部汁液中的钠含量降低。与Na含量相比，athkt1突变等位基因对木质部K含量的影响较小且相反，这表明K的运输可能受到间接影响。AtHKT1的表达不仅受钠、钾浓度的调节，还受非离子化合物渗透压的调节。M2D节段中部附近保守的Arg对于KtrB的K~+转运是必不可少的。我们提出在M2D中的正残基和孔区保守的负残基之间存在盐桥(S)，它降低了正残基(S)对阳离子渗透的静电屏障，从而稳定了转运蛋白的构型。KtrB第二孔环中保守的His-157突变使聚球藻PCC6803的K~+转运蛋白KtrABE系统活性显著降低。这一结果提示His-157在转运蛋白系统的K~+转运活动中起着重要作用。利用表达聚球藻Na/H转运蛋白的倒置膜泡，测定了该蛋白的H转运活性。我们已经确定了钾运输所必需的带电残基。

1. We have identified the localization of Arabidopsis Na transporter AtHKT1 in plant cells. The specificity of the anti-AtHKT1 antibodies was confirmed. Immunoelectron microscopy using the antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves.2. AtHKT1 disruption alleles caused large increases in the Na content of the xylem sap and conversely reduced the Na content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the K content compared with the Na content to the xylem, suggesting that K transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na and K but also by the osmolality of non-ionic compounds.3. The conserved Arg near the middle of the M2D segment was essential for the K^+ transport of KtrB. We propose the existence of the salt bridge(s) between the positive residues in the M2D and the conserved negative residues in the pore region, which reduces an electrostatic barrier against cation permeation caused by the positive residue(s) and that stabilizes the transporter configuration.4. Mutation of a conserved His-157 in the second pore loop of KtrB, drastically reduced the activity of the K^+ transporter KtrABE system from Synechocystis PCC 6803. The result suggests that His-157 plays an essential role in the K^+ transport activity of the transporter system.5. H transport activity of one of Synechocystis Na/H transporters was measured using the inverted membrane vesicle of E. coli expressing the protein. We have identified the charged residues essential for the K transport.