An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells.
An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells.
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DOI：
10.1111/tpj.12257
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发表时间：
2013-09
期刊：
影响因子：
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通讯作者：
Jinbo Shen;Pui Kit Suen;Xiangfeng Wang;Youshun Lin;Sze Wan Lo;E. Rojo;Liwen Jiang
中科院分区：
文献类型：
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作者：
Jinbo Shen;Pui Kit Suen;Xiangfeng Wang;Youshun Lin;Sze Wan Lo;E. Rojo;Liwen Jiang
液泡分选受体（vrs）是一类完整的膜家族蛋白，在植物细胞中被认为识别晚期高尔基或反式高尔基网络（TGN）中的货物蛋白，通过液泡前室（PVC）进行液泡运输。然而，对植物中的VSR货物蛋白知之甚少。在此，我们开发并测试了一种鉴定VSR载体的体内表达系统，该系统的前提是表达的VSR的n端会与其相应的载体蛋白一起分泌到培养基中。事实上，表达VSR n末端结合域（VSRNTs）的转基因拟南芥培养细胞系被证明可以分泌截断的VSR (BP80NT， AtVSR1NT和AtVSR4NT)，并附着货物分子到培养基中。假定的货物蛋白通过质谱鉴定。通过定位研究和与VSRs的相互作用分析，证实了几种鉴定的货物蛋白。本文描述的筛选策略应适用于所有VSR，并将有助于识别和研究单个VSR蛋白的货物蛋白。该方法可用于货物鉴定和体内蛋白质相互作用。
Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the pre-vacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here we developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture medium along with their corresponding cargo proteins. Indeed, transgenic Arabidopsis culture cell lines expressing VSR N-terminal binding domains (VSRNTs) were shown to secrete truncated VSRs (BP80NT, AtVSR1NT and AtVSR4NT) with attached cargo molecules into the culture medium. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by localization studies and interaction analysis with VSRs. The screening strategy described here should be applicable to all VSRs and will help identify and study cargo proteins for individual VSR proteins. This method should be useful for both cargo identification and protein-protein interaction in vivo.