Rombolá-Caldentey Belén, Mendoza Imelda, Quintero Francisco J, Pardo José M
Instituto de Bioquimica Vegetal y Fotosintesis, cicCartuja, Consejo Superior de Investigaciones Cientificas, Universidad de Sevilla, 41092 Sevilla, Spain.
Plants (Basel). 2023 Jul 26;12(15):2778. doi: 10.3390/plants12152778.
Cation/Proton Antiporters (CPA) acting in all biological membranes regulate the volume and pH of cells and of intracellular organelles. A key issue with these proteins is their structure-function relationships since they present intrinsic regulatory features that rely on structural determinants, including pH sensitivity and the stoichiometry of ion exchange. Crystal structures are only available for prokaryotic CPA, whereas the eukaryotic ones have been modeled using the former as templates. Here, we present an updated and improved structural model of the tonoplast-localized K, Na/H antiporter NHX1 of Arabidopsis as a representative of the vacuolar NHX family that is essential for the accumulation of K into plant vacuoles. Conserved residues that were judged as functionally important were mutated, and the resulting protein variants were tested for activity in the yeast . The results indicate that residue N184 in the ND-motif characteristic of CPA1 could be replaced by the DD-motif of CPA2 family members with minimal consequences for their activity. Attempts to alter the electroneutrality of AtNHX1 by different combinations of amino acid replacements at N184, R353 and R390 residues resulted in inactive or partly active proteins with a differential ability to control the vacuolar pH of the yeast.
阳离子/质子反向转运蛋白（CPA）存在于所有生物膜中，可调节细胞及细胞内细胞器的体积和pH值。这些蛋白质的一个关键问题是它们的结构-功能关系，因为它们具有依赖于结构决定因素的内在调节特性，包括pH敏感性和离子交换的化学计量。目前仅有原核CPA的晶体结构，而真核CPA则以前者为模板进行建模。在此，我们展示了拟南芥液泡膜定位的钾、钠/氢反向转运蛋白NHX1的更新且改进的结构模型，它是液泡NHX家族的代表，对钾在植物液泡中的积累至关重要。将被判定为功能重要的保守残基进行突变，并对产生的蛋白质变体在酵母中的活性进行测试。结果表明，CPA1特征性ND基序中的N184残基可被CPA2家族成员的DD基序取代，对其活性影响最小。通过在N184、R353和R390残基处进行不同氨基酸替换组合来改变AtNHX1的电中性的尝试，产生了无活性或部分活性的蛋白质，它们控制酵母液泡pH值的能力有所不同。
