Subcellular localisation of an endoplasmic reticulum-plasma membrane tethering factor, SYNAPTOTAGMIN 1, is affected by fluorescent protein fusion
Subcellular localisation of an endoplasmic reticulum-plasma membrane tethering factor, SYNAPTOTAGMIN 1, is affected by fluorescent protein fusion
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内质网质膜束缚因子 SYNAPTOTAGMIN 1 的亚细胞定位受荧光蛋白融合的影响
DOI：
10.1080/15592324.2018.1547577
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发表时间：
2019
影响因子：
2.9
通讯作者：
and T. Shimada
中科院分区：
文献类型：
--
作者：
K. Ishikawa;Kentaro Tamura;and T. Shimada
膜接触位点（MCS）由于其在许多重要的细胞过程中的普遍作用而日益受到关注。SYT 1是植物细胞中连接内质网（ER）和质膜（PM）的一种束缚因子。使用荧光蛋白融合的共聚焦显微镜是研究蛋白质定位和功能的不可或缺的工具。然而，几项研究报道，荧光蛋白二聚化影响荧光蛋白标记的蛋白质的亚细胞定位。在这里，我们调查的荧光蛋白二聚化的影响，通过比较SYT 1与合成的GFP融合（SYT 1-sGFP）和SYT 1与单体GFP融合（SYT 1-mGFP）的亚细胞定位。SYT 1-mGFP局限于ER中的特定结构域，而SYT 1-sGFP在瞬时过表达时沿ER扩散沿着。SYT 1-本地化的区域，建议对应ER-PM接触网站，因为它的固定性。在转基因拟南芥中获得了类似的结果，即使SYT 1-sGFP和SYT 1-mGFP的表达水平相当。这表明，SYT 1-mGFP更准确地再现SYT 1定位在完整的细胞，因为在ER中的持久区域的比例是更相似的野生型和表达SYT 1-mGFP的植物之间比野生型和表达SYT 1-sGFP的植物。综上所述，这些结果表明，sGFP的融合使SYT 1-sGFP在ER中形成过多的ER-PM接触位点。
Membrane contact sites (MCS) have increasingly received attention because of their general role in a number of important cellular processes. SYNAPTOTAGMIN 1 (SYT1) is a tethering factor connecting the endoplasmic reticulum (ER) and the plasma membrane (PM) in plant cells. Confocal microscopy using fluorescent protein fusion is an indispensable tool for studying protein localisation and functions. However, several studies have reported that fluorescent protein dimerisation affects the subcellular localisation of proteins tagged by the fluorescent protein. Here, we investigate the effects of fluorescent protein dimerisation by comparing the subcellular localisation of SYT1 fused with a synthetic GFP (SYT1-sGFP) and SYT1 fused with a monomeric GFP (SYT1-mGFP). SYT1-mGFP was confined to specific domains in the ER, whereas SYT1-sGFP spread along the ER when transiently overexpressed. SYT1-localised regions were suggested to correspond to ER-PM contact sites because of its immobility. Similar results were obtained in the transgenic Arabidopsis, even though SYT1-sGFP and SYT1-mGFP were expressed at comparable levels. It is suggested that SYT1-mGFP more accurately reproduced SYT1 localisation in intact cells because the proportion of persistent area in the ER was more similar between the wild type and the plant expressing SYT1-mGFP than between the wild type and the plant expressing SYT1-sGFP. Taken together, these results suggest that the fusion of sGFP makes SYT1-sGFP form excessive ER-PM contact sites in the ER.