Generation of Arabidopsis lines with a red fluorescent marker for endoplasmic reticulum using a tail-anchored protein cytochrome b5-B. (1st September 2020)

• Record Type: Journal Article
• Authors: Nagano, Minoru; Ueda, Haruko; Fukao, Yoichiro; Kawai-Yamada, Maki; Hara-Nishimura, Ikuko
• Is Part Of: Plant signaling & behavior. Volume 15:Number 9(2020)
• Publication Date: 2020-09-01
• Subjects: Endoplasmic reticulum -- red fluorescent protein -- tail-anchored protein -- cytochrome b5; Plant ecophysiology -- Periodicals; Plant cellular signal transduction -- Periodicals; Plant cellular signal transduction; Plant ecophysiology; Periodicals; Electronic journals; 581
• DOI: 10.1080/15592324.2020.1790196
• Languages: English
• ISSNs: 1559-2316

Abstract

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.