定位于植物内质网-质膜接触位点的蛋白质的表征
Characterization of Proteins Localized to Plant ER-PM Contact Sites.
作者信息
Wang Pengwei, Hawes Chris, Richardson Christine, Hussey Patrick J
机构信息
Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, Hubei Province, 430070, PR China.
Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, OX3 0BP, UK.
出版信息
Methods Mol Biol. 2018;1691:23-31. doi: 10.1007/978-1-4939-7389-7_3.
Like in most eukaryotic cells, the plant endoplasmic reticulum (ER) network is physically linked to the plasma membrane (PM), forming ER-PM contact sites (EPCS). The protein complex required for maintaining the EPCS is composed of ER integral membrane proteins (e.g., VAP27, synaptotagmins), PM-associated proteins (e.g., NET3C), and the cytoskeleton. Here, we describe methods for identifying possible EPCS-associated proteins. These include GFP-tagged protein expression followed by image analysis, and immuno-gold labeling at the ultrastructural level. In combination, these methods can be used to identify the localization of putative EPCS proteins as well as used to postulate their subcellular function.
与大多数真核细胞一样，植物内质网（ER）网络与质膜（PM）物理相连，形成内质网 - 质膜接触位点（EPCS）。维持EPCS所需的蛋白质复合物由内质网整合膜蛋白（如VAP27、突触结合蛋白）、质膜相关蛋白（如NET3C）和细胞骨架组成。在此，我们描述了鉴定可能与EPCS相关的蛋白质的方法。这些方法包括表达绿色荧光蛋白标记的蛋白质，随后进行图像分析，以及在超微结构水平进行免疫金标记。综合起来，这些方法可用于确定假定的EPCS蛋白的定位，并推测其亚细胞功能。
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