Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization
Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization
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DOI：
10.1073/pnas.1301160110
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发表时间：
2013-07
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影响因子：
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通讯作者：
Qinli Wang;Yuanyuan Zhao;Wangxi Luo;Ruili Li;Qi-hua He;X. Fang;R. Michele;C. Ast;N. von Wirén-N.-vo
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文献类型：
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作者：
Qinli Wang;Yuanyuan Zhao;Wangxi Luo;Ruili Li;Qi-hua He;X. Fang;R. Michele;C. Ast;N. von Wirén-N.-vo
铵是植物的优选氮源，但在高水平下是有毒的。植物铵转运蛋白（AMTs）在NH 4+吸收过程中起着重要作用，但AMTs的调控机制尚不清楚。为了研究AMTs是如何在铵的存在下进行调节的，我们使用可变角度全内反射荧光显微镜和荧光互相关光谱法对不同铵水平下拟南芥根细胞质膜上的EGFP标记的AMT 1;3进行单粒子荧光成像。我们证明了AMT 1;在缺氮和氮充足条件下，3-EGFP以低寡聚态的移动荧光点在质膜上动态出现和消失。在高铵胁迫下，AMT 1;3-EGFPs聚集成簇，然后内化到细胞质中。类似的现象也发生在谷氨酰胺合成酶突变体gln 1;2背景中。网格蛋白重链2突变体中AMT 1;3-EGFPs的单颗粒分析（chc 2突变体）和Flotlin 1人工microRNA（Flot 1 amiRNA）背景，以及化学抑制剂处理，证明了AMT 1的内吞作用; 3高铵胁迫诱导的聚集主要通过网格蛋白介导的内吞途径，但不能排除微区相关的内吞途径在内化中的作用。我们的研究结果表明，AMT 1;3的聚集和内吞作用提供了一种有效的机制，植物细胞可以通过消除质膜上的活性AMT 1;3来避免有毒水平的铵的积累。
Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH4+ uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane.