![]() It is not known whether the properties of RTNLB13, which contains the conserved RHD but has small soluble N- and C-terminal domains and is predicted to be expressed in seeds (see Supplemental Figure 1 online Winter et al., 2007), are shared by the more ubiquitously expressed members of the RTN family, such as RTNLB1, 2, 3, and 4 (see Supplemental Figure 1 online). The ability of a surfeit of RTN to cause constrictions fits the hypothesis that RTNs not only individually bend the membrane but can also form multimeric, arc-like structures ( Shibata et al., 2008, 2009), which may ultimately shape and determine the diameter of ER tubules ( Hu et al., 2008). These constrictions reduce diffusion within the ER lumen but do not cause major anterograde (from the ER toward the Golgi) trafficking defects. We have recently shown that the smallest plant reticulon isoform, RTNLB13, localizes to the ER membrane and, when overexpressed, can dramatically affect ER morphology by causing constrictions in the ER tubules ( Tolley et al., 2008). RTNLB (for Reticulon Like Protein subfamily B) 1, 2, and 4 were isolated in a screen for agrobacterial pilin-interacting proteins, and a direct correlation between the level of RTNLB1 and plant susceptibility to Agrobacterium tumefaciens transformation was documented ( Hwang and Gelvin, 2004). However, very little information is available regarding plant RTNs and their potential functions. This suggests that the numerous RTN isoforms may perform plant-specific functions. The RTN gene family is much larger in higher plants than it is in mammals, with Arabidopsis thaliana having 21 RTN genes ( Oertle et al., 2003 Nziengui et al., 2007 Nziengui and Schoefs, 2009 Sparkes et al., 2009b). It is assumed that these curvature-inducing properties result from the wedge-like transmembrane topology of the conserved structural motif of these proteins (the reticulon homology domain ), which includes two large hydropobic segments ( Shibata et al., 2009). Recently, a class of ER membrane proteins named the reticulons (RTNs) was shown to be sufficient to induce ER tubulation in yeast cells, mammalian cells ( Voeltz et al., 2006), and in artificial proteoliposomes ( Hu et al., 2008). This shape is independent of the ER being attached to a functional cytoskeleton ( Boevink et al., 1998 Dreier and Rapoport, 2000), indicating that factors within the membrane itself must be responsible for conferring its curvature. In cells of plant vegetative tissues, the cortical ER is predominantly tubular with small cisternal patches. The plant ER is an extremely dynamic organelle with a unique architecture consisting of a network of membrane tubules and sheets (cisternae) connected by three-way junctions (for a recent review, see Sparkes et al., 2009b). The endoplasmic reticulum (ER) is the site of synthesis for many cellular proteins and the port of entry into the secretory pathway. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions. ![]() We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Reticulons are integral membrane proteins involved in shaping ER tubules. The cortical endoplasmic reticulum (ER) in tobacco ( Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. ![]()
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