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.
Keywords: Chi-square analysis; ER-PM contact sites; Endoplasmic reticulum; Immuno-gold TEM; N. benthamiana; NET3C; VAP27 family.