ABSTRACT 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 cytochrome b5 -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, and Nicotiana benthamiana. We believe that TA proteins could be used to generate various organellar membrane markers in plants.

中文翻译：

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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


摘要 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、水稻和本塞姆氏烟草的 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。