Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis

Abstract

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.

Modeling Endoplasmic Reticulum Network Maintenance in a Plant Cell

“…5 predict that either faster cross-connection or slower tension drift will give statistically smaller polygons. In principle, this prediction can be experimentally tested by analysis of the ER when manipulating molecular factors that control ER geometry and dynamics (2,8,9,(29)(30)(31)(32)(33)(34)(35)(36). A more sophisticated model will take other biophysical processes into account.…”

Advances in Plant ER Architecture and Dynamics

“…Loss of function of RHD3 leads to a reduced elongation of cells of the primary root (Stefano et al, 2012). At a subcellular level, the loss of RHD3 also reduces the formation of three-way junctions owing to the capacity of the protein to fuse membranes in a GTP-dependent fashion (Ueda et al, 2016). In this context, RHD3 functions analogously to similar membrane-associated dynamin-like GTPases, such as metazoan atlastin and yeast Sey1p Zhang et al, 2013;Yan et al, 2015).…”

“…In particular, overexpression of the C-terminal region of RHD3 (RHD3 amino acids 677-802) disrupts the ER network integrity; conversely, overexpression of the analogous Sey1p region (Sey1p amino acids 682-776) does not . Intriguingly, the C-terminal domain of RHD3 is phosphorylated, and it has been shown that kinase treatment of RHD3 induces oligomerization of this protein, which in turn may modulate its ER-shaping function (Ueda et al, 2016).…”

The calcium‐binding protein ALG‐2 promotes endoplasmic reticulum exit site localization and polymerization of Trk‐fused gene (TFG) protein

“…Samples were analyzed by nano‐flow reverse phase liquid chromatography followed by tandem MS, using a Triple TOF 5600+, essentially described as previously . Briefly, a capillary reverse phase HPLC‐MS/MS system composed of an Eksigent Ekspert nano‐LC 400 HPLC system (AB Sciex), which was directly connected to an AB Sciex quadrupole time‐of‐flight (QqTOF) TripleTOF 5600+ mass spectrometer in Trap and Elute mode.…”