A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon.

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.

Long-Term Imaging of Endoplasmic Reticulum Morphology in Embryos During Seed Germination.

Imaging plant embryos at the cellular level over time is technically challenging, since the embryo, once its protective seed coat is removed, must be kept viable and unstressed on a microscope slide for the duration of the experiment. Here we describe a procedure and suitable apparatus for the visualization, over several days, of changes in endoplasmic reticulum (ER) morphology associated with the process of germination in Arabidopsis thaliana seeds. Moreover, we also present a user-friendly image analysis tool, which enables subtle perturbations in the ER network to be measured.

An Arabidopsis reticulon and the atlastin homologue RHD3-like2 act together in shaping the tubular endoplasmic reticulum.

The endoplasmic reticulum (ER) is a network of membrane sheets and tubules connected via three-way junctions. A family of proteins, the reticulons, are responsible for shaping the tubular ER. Reticulons interact with other tubule-forming proteins (Dp1 and Yop1p) and the GTPase atlastin. The Arabidopsis homologue of Dp1/Yop1p is HVA22. We show here that a seed-specific isoform of HVA22 labels the ER in tobacco (Nicotiana tabacum) cells but its overexpression does not alter ER morphology. The closest plant homologue of atlastin is RHD3. We show that RHD3-like 2 (RL2), the seed-specific isoform of RHD3, locates to the ER without affecting its shape or Golgi mobility. Expression of RL2-bearing mutations within its GTPase domain induces the formation of large ER strands, suggesting that a functional GTPase domain is important for the formation of three-way junctions. Coexpression of the reticulon RTNLB13 with RL2 resulted in a dramatic alteration of the ER network. This alteration did not depend on an active GTPase domain but required a functional reticulon, as no effect on ER morphology was seen when RL2 was coexpressed with a nonfunctional RTNLB13. RL2 and its GTPase mutants coimmunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 act together in modulating ER morphology.

Long-Term Imaging of Endoplasmic Reticulum Morphology in Embryos During Seed Germination.

Imaging plant embryos at the cellular level over time is technically challenging, since the embryo, once its protective seed coat is removed, must be kept viable and unstressed on a microscope slide for the duration of the experiment. Here we describe a procedure and suitable apparatus for the visualization, over several days, of changes in endoplasmic reticulum (ER) morphology associated with the process of germination in Arabidopsis thaliana seeds. Moreover, we also present a user-friendly image analysis tool which enables subtle perturbations in the ER network to be measured.