Five Arabidopsis Reticulon Isoforms Share Endoplasmic Reticulum Location, Topology, and Membrane-Shaping Properties

Abstract

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Fö rster 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. 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.

The reticulons: Guardians of the structure and function of the endoplasmic reticulum

“…Consistent with this hypothesis, in Arabidopsis thaliana it was demonstrated that the lengths of the transmembrane segments in RTNLB13 are likely to be responsible for its ER membrane curvature [8]. Subsequently, together with RTNLB13, four other members of the reticulon family, RTNLB1-4, have shown a similar effect on the ER structure in A. thaliana [9].…”

Arabidopsis Lunapark proteins are involved in ER cisternae formation

“…Another class of proteins known to be required for the formation of a dynamic ER tubular network in plants are the reticulon (RTN) proteins, which induce and/or stabilize membrane curvature and are capable of constricting tubules whilst suppressing cisternae formation (Sparkes et al ., 2010). Hence, we explored the potential interplay between RTN and LNP proteins.…”

“…mRFP) that is independent of the problems associated with steady‐state intensity measurements. Reduction in GFP lifetime is an indication that the two proteins are within a distance of 1–10 nm, thus indicating a direct physical interaction between the two protein fusions (Sparkes et al ., 2010; Schoberer & Botchway, 2014). It was previously shown that a reduction of as little as 200 ps in the excited‐state lifetime of the GFP‐labelled protein represents quenching through a protein–protein interaction (Stubbs et al ., 2005).…”

“…AtLNP1 and AtLNP2 constructs were expressed using an OD 600 of 0.3 that usually results in the formation of enlarged cisternae (Fig. 4), RTN1 was infiltrated at a standard OD 600 of 0.1 known to result in ER tubule restriction (Sparkes et al ., 2010). Transient coexpression of AtLNP1‐GFP with RFP‐RTN1 (Fig.…”

“…One hypothetical scenario could be that LNP proteins which are labelling cisternae but not the edges (Fig. 3c,d) induce the flat membranes of cisternae, whereas reticulons that localize to the edge of cisternae (Sparkes et al ., 2010) induce curvature, thereby limiting cisternal expansion. In contrast to that described for their yeast and mammalian orthologues, Arabidopsis LNP proteins label only a small proportion of junctions which could also potentially be small cisternae, and we did not find any evidence that they stabilize ER three‐way junctions.…”