Abstract
ROOT HAIR DEFECTIVE3 (RHD3) is an atlastin GTPase involved in homotypic fusion of endoplasmic reticulum (ER) tubules in the formation of the interconnected ER network. Because excessive fusion of ER tubules will lead to the formation of sheet-like ER, the action of atlastin GTPases must be tightly regulated. We show here that RHD3 physically interacts with two Arabidopsis (Arabidopsis thaliana) LUNAPARK proteins, LNP1 and LNP2, at three-way junctions of the ER, the sites where different ER tubules fuse. Recruited by RHD3 to newly formed three-way junctions, LNPs act negatively with RHD3 to stabilize the nascent three-way junctions of the ER. Without this LNP-mediated stabilization, in Arabidopsis lnp1-1 lnp2-1 mutant cells, the ER becomes a dense tubular network. Interestingly, in lnp1-1 lnp2-1 mutant cells, the expression level of RHD3 is higher than that in wild-type plants. RHD3 is degraded more slowly in the absence of LNPs as well as in the presence of MG132 and concanamycin A. However, in the presence of LNPs, the degradation of RHD3 is promoted. We have provided in vitro evidence that Arabidopsis LNPs have E3 ubiquitin ligase activity and that LNP1 can directly ubiquitinate RHD3. Our data show that after ER fusion is completed, RHD3 is degraded by LNPs so that nascent three-way junctions can be stabilized and a tubular ER network can be maintained.