The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.
The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.
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DOI：
10.3389/fpls.2016.00862
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发表时间：
2016
影响因子：
5.6
通讯作者：
Johanson U
中科院分区：
文献类型：
--
作者：
Ampah-Korsah H;Anderberg HI;Engfors A;Kirscht A;Norden K;Kjellstrom S;Kjellbom P;Johanson U
水通道蛋白（AQP）是一种跨膜转运水和不带电溶质的膜通道蛋白。在植物中，水通道蛋白可分为七个不同的亚家族，其中五个存在于高等植物中。这些亚科中最新的特征是XIP亚科，它在大多数双子叶植物中发现，但在单子叶植物中没有。在这篇文章中，我们提出了两个不同的剪接变异体（α和β）的NbXIP 1;1从烟草本塞姆亚纳。我们描述了NbXIP 1;1α和β在毕赤酵母中的异源表达、该蛋白在该系统中的亚细胞定位以及NbXIP 1;1α蛋白的纯化。此外，我们通过停流光谱法在巴斯德毕赤酵母原生质球中以及在蛋白脂质体中重构的蛋白质研究了蛋白质的功能性和底物特异性。通过质谱法验证蛋白质的磷酸化状态和磷酸化氨基酸的定位。我们的研究结果表明，NbXIP 1;1α在巴斯德毕赤酵母中表达时位于质膜上，它不透水，但透硼酸，并且该蛋白质在N-末端胞质结构域的几个氨基酸处被磷酸化。生长测定显示，与表达C-末端His-标记的同种型的细胞相比，表达N-末端His-标记的NbXIP 1;1α的酵母细胞对硼酸更敏感。这可能表明N-末端His-标签在功能上模拟N-末端结构域的磷酸化，并且N-末端结构域参与通道的门控。
Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.