Efficient ER Fusion Requires a Dimerization and a C-Terminal Tail Mediated Membrane Anchoring of RHD3

The endoplasmic reticulum (ER) is a network of tubules and sheets stretching throughout the eukaryotic cells. The formation of the ER requires homotypic membrane fusion, which is mediated by a family of Dynamin-like Atlastin GTPase proteins. The Arabidopsis (Arabidopsis thaliana) member ROOT HAIR DEFECTIVE3 (RHD3) has been demonstrated to mediate ER membrane fusion, but how exactly RHD3 is involved in the process is still unknown. Here we conducted systemic structure-function analyses of roles of different RHD3 domains in mediating ER fusion. We showed that efficient ER membrane fusion mediated by RHD3 requires a proper dimerization of RHD3 through the GTPase domain (GD) and the first and second three helix bundles (3HBs) in the middle domain. RHD3 has a 3HB-enriched middle domain longer than that of Atlastins, and we revealed that the third and fourth 3HBs are required for the stability of RHD3. The transmembrane segments of RHD3 are essential for targeting and retention of RHD3 in the ER and can also facilitate an oligomerization of RHD3. Furthermore, we showed that an amphipathic helix in the C-terminal cytosolic tail of RHD3 has a membrane anchoring ability that is required for efficient ER membrane fusion mediated by RHD3. This work contributes to a better understanding of a coordinated action of RHD3 in the fusion of ER membranes.