Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata,Endoplasmic Reticulum,and the Plasma Membrane

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane.The endoplasmic reticulum (ER) is a multifunctional organelle (Hawes et al., 2015) and is the site of secretory protein production, folding, and quality control (Brandizzi et al., 2003) and lipid biosynthesis (Wallis and Browse, 2010), but it is also involved in many other aspects of day-to-day plant life, including auxin regulation (Friml and Jones, 2010) and oil and protein body formation (Huang, 1996; Herman, 2008). The cortical ER network displays a remarkable polygonal arrangement of motile tubules that are capable of morphing into small cisternae, mainly at the three-way junctions of the ER network (Sparkes et al., 2009). The cortical ER network of plants has been shown to play multiple roles in protein trafficking (Palade, 1975; Vitale and Denecke, 1999) and pathogen responses (for review, see Pattison and Amtmann, 2009; Beck et al., 2012).In plants, the protein family of reticulons (RTNLBs) contributes significantly to tubulation of the ER (Tolley et al., 2008, 2010; Chen et al., 2012). RTNLBs are integral ER membrane proteins that feature a C-terminal reticulon homology domain (RHD) that contains two major hydrophobic regions. These regions form two V-shaped transmembrane wedges joined together via a cytosolic loop, with the C and N termini of the protein facing the cytosol. RTNLBs can dimerize or oligomerize, creating localized tensions in the ER membrane, inducing varying degrees of membrane curvature (Sparkes et al., 2010). Hence, RTNLBs are considered to be essential in maintaining the tubular ER network.The ability of RTNLBs to constrict membranes is of interest in the context of cell plate development and the formation of primary plasmodesmata (PD; Knox et al., 2015). PD formation involves extensive remodeling of the cortical ER into tightly furled tubules to form the desmotubules, axial structures that run through the PD pore (Overall and Blackman, 1996; Ehlers and Kollmann, 2001). At only 15 nm in diameter, the desmotubule is one of the most constricted membrane structures found in nature, with no animal counterparts (Tilsner et al., 2011). PD are membrane-rich structures characterized by a close association of the plasma membrane (PM) with the ER. The forces that model the ER into desmotubules, however, are poorly understood. RTNLBs are excellent candidates for this process and can constrict fluorescent protein-labeled ER membranes into extremely fine tubules (Sparkes et al., 2010). We showed recently that two of the RTNLBs present in the PD proteome, RTNLB3 and RTNLB6 (Fernandez-Calvino et al., 2011), are present in primary PD at cytokinesis (Knox et al., 2015). However, nothing is known of the proteins that interact with RTNLBs identified in the PD proteome or that may link RTNLBs to the PM. To date, the only protein shown to bind to plant RTNLBs is RHD3-LIKE2, the plant homolog of the ER tubule fusion protein ATLASTIN (Lee et al., 2013).Here, we used a dual approach to identify interacting partners of RTNLB3 and RTNLB6 (Fernandez-Calvino et al., 2011; Knox et al.., 2015). First, we used GFP immunoprecipitation assays coupled to mass spectrometry (MS) to identify proteins potentially binding to RTNLB3 and RTNLB6. Second, from the proteins we identified, we conducted a detailed Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FRET-FLIM) analysis to confirm prey-bait interactions in vivo.The application of time-resolved fluorescence spectroscopy to imaging biological systems has allowed the design and implementation of fluorescence lifetime imaging microscopy (FLIM). The technique allows measuring and determining the space map of picosecond fluorescence decay at each pixel of the image through confocal single and multiphoton excitation. The general fluorescence or Förster resonance energy transfer (FRET) to determine the colocalization of two color chromophores can now be improved to determine physical interactions using FRET-FLIM and protein pairs tagged with appropriate GFP fluorophores and monomeric red fluorescent protein (mRFP). FRET-FLIM measures the reduction in the excited-state lifetime of GFP (donor) fluorescence in the presence of an acceptor fluorophore (e.g. mRFP) that is independent of the problems associated with steady-state intensity measurements. The observation of such a reduction is an indication that the two proteins are within a distance of 1 to 10 nm, thus indicating a direct physical interaction between the two protein fusions (Osterrieder et al., 2009; Sparkes et al., 2010; Schoberer and Botchway, 2014). It was shown previously that a reduction of as little as approximately 200 ps in the excited-state lifetime of the GFP-labeled protein represents quenching through a protein-protein interaction (Stubbs et al., 2005).Our interaction data identified a large percentage (40%) of ER proteins, including other RTNLB family members. However, we also found a relatively large number (25%) of proteins present in the published PD proteome (Fernandez-Calvino et al., 2011) and a surprisingly high proportion (35%) of PM proteins. Of the PD-resident proteins we identified, a significant number were shown previously to be targets of viral movement proteins (MPs) or proteins present within lipid rafts, consistent with the view that PD are lipid-rich microdomains (Bayer et al., 2014). Additional proteins identified suggested roles for RTNLBs in transport and pathogen defense. We suggest that RTNLBs may play key roles in anchoring and/or signaling between the cortical ER and PM.