Phosphorylation of the C-terminus of RHD3 Has a Critical Role in 21 Homotypic ER Membrane Fusion in Arabidopsis 1[W]

One-sentence summary: The Arabidopsis dynamin-like GTPase RHD3 has membrane fusion activity needed to form a proper endoplasmic reticulum network and this activity is regulated by phosphorylation of its C-terminus. ABSTRACT 63 The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In 64 animals and yeast, homotypic ER membrane fusion is mediated by fusogens (Atlastin and 65 Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis 66 thaliana , another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been 67 proposed as an ER membrane fusogen but direct evidence is lacking. Here, we show that 68 RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its 69 C-terminus. The ER network was RHD3-dependently reconstituted from the 70 cytosol-and-microsome fraction of tobacco cultured cells by exogenously adding GTP, ATP, 71 and F-actin. We next established an in-vitro assay system of ER tubule formation with A. 72 thaliana ER vesicles, in which addition of GTP caused ER sac formation from the ER 73 vesicles. Subsequent application of a shearing force to this system triggered the formation 74 of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence 75 of a shearing force, serine/threonine-kinase treatment triggered RHD3-dependent tubule 76 formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple serine 77 and threonine residues in the C-terminus. An antibody against the RHD3 C-terminal 78 peptide abolished kinase-triggered tubule formation. When the serine cluster was deleted 79 or when the serine residues were replaced with alanine residues, kinase treatment had no 80 effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither 81 phosphorylation-dependent modulation of membrane fusion nor oligomerization have been 82 reported for Atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated 83 oligomerization of RHD3 enhances ER membrane fusion to form the ER network. assay system ER tubule elongation ER vesicles nucleotides shearing formation assay. The cytosol-microsome fraction (S12 fraction) from cultured 757 tobacco cells expressing ER-luminal GFP was incubated for ~70 min in the absence (mock 758 in A) or in the presence of actin filaments (F-actin) and the nucleotides GTP and ATP (B) 759 and then was inspected with a confocal laser scanning microscope. Note that the ER 760 tubules and cisternae were efficiently generated from the ER microsomes by incubating 761 with F-actin and the nucleotides, resulting in creating the ER network in vitro . C and D, An 762 S12 fraction that was pre-incubated with either anti-RHD3NT (C) or control IgG (D) was 763 subjected to the in-vitro ER network formation assay. Note that anti-RHD3NT, but not 764 control IgG, abolished the formation of ER tubules and cisternae, which prevented ER 765 network formation. E and F, Quantitative analyzes of ER cisterna formation (E) and ER 766 tubule formation (F) during incubation of ER microsomes with F-actin and nucleotides. The 767 areas of ER cisternae and the lengths of ER tubules were calculated by MorphoER 768 software at the indicated time points. The experiments were repeated at least three times.