Generation of Arabidopsis lines with a red fluorescent marker for endoplasmic reticulum using a tail-anchored protein cytochrome b5 -B
Generation of Arabidopsis lines with a red fluorescent marker for endoplasmic reticulum using a tail-anchored protein cytochrome b5 -B
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使用尾部锚定蛋白细胞色素 b5 -B 生成具有内质网红色荧光标记的拟南芥品系
DOI：
10.1080/15592324.2020.1790196
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发表时间：
2020
影响因子：
2.9
通讯作者：
Hara-Nishimura Ikuko
中科院分区：
文献类型：
--
作者：
Nagano Minoru;Ueda Haruko;Fukao Yoichiro;Kawai-Yamada Maki;Hara-Nishimura Ikuko
内质网（ER）是真核生物中执行多种细胞活动的多功能细胞器。使用荧光蛋白可视化 ER 是分析其动态和了解其功能的有效方法。然而，同时具有 N 端信号肽 (SP) 和 C 端 ER 保留四肽 (HDEL) 的红色荧光蛋白在拟南芥中组成型表达时，通常会导致错误定位到液泡或细胞外空间。为了获得红色荧光 ER 标记，我们选择了拟南芥细胞色素 b5-B (Cb5-B)，一种 ER 膜上的尾锚定 (TA) 蛋白。其定位由 C 末端的跨膜结构域 (TMD) 和尾部结构域决定。我们将 TMD 和 Cb5-B 的尾部结构域融合到红色荧光蛋白 tdTomato (tdTomato-CTT) 的 C 末端。当 tdTomato-CTT 在拟南芥中的泛素 10 启动子下组成型表达时，通过可靠的 ER 标记 SP-GFP-HDEL 只能在 ER 处检测到荧光信号。因此，tdTomato-CTT 可以准确地可视化稳定拟南芥品系中的 ER。此外，瞬时测定表明 tdTomato-CTT 还可用作洋葱、水稻和烟草的 ER 标记。我们相信 TA 蛋白可用于在植物中产生各种细胞器膜标记。
The endoplasmic reticulum (ER) is a multifunctional organelle that performs multiple cellular activities in eukaryotes. Visualizing ER using fluorescent proteins is a powerful method of analyzing its dynamics and to understand its functions. However, red fluorescent proteins with both an N-terminal signal peptide (SP) and a C-terminal ER retention tetrapeptide (HDEL) often cause mislocalization to vacuoles or extracellular spaces when they are constitutively expressed in Arabidopsis. To obtain a red fluorescent ER marker, we selected Arabidopsis cytochromeb5-B (Cb5-B), a tail-anchored (TA) protein on the ER membrane. Its localization is determined by the transmembrane domain (TMD) and tail domain at the C-terminus. We fused the TMD and the tail domain of Cb5-B to the C-terminus of a red fluorescent protein, tdTomato (tdTomato-CTT). When tdTomato-CTT was constitutively expressed under the ubiquitin10 promoter in Arabidopsis, the fluorescent signal was exclusively detected at the ER by means of the reliable ER marker SP-GFP-HDEL. Therefore, tdTomato-CTT can accurately visualize the ER in stable Arabidopsis lines. Additionally, transient assays showed that tdTomato-CTT can also be used as an ER marker in onion, rice, andNicotiana benthamiana. We believe that TA proteins could be used to generate various organellar membrane markers in plants.