Bian, X. et al. Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes. Proc. Natl Acad. Sci. USA 108, 3976-3981

Department of Genetics and Cell Biology, College of Life Sciences, and Tianjin Key Laboratory of Protein Sciences, Nankai University, Tianjin 300071, China.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2011; 108(10):3976-81. DOI: 10.1073/pnas.1101643108
Source: PubMed


The generation of the tubular network of the endoplasmic reticulum (ER) requires homotypic membrane fusion that is mediated by the dynamin-like, membrane-bound GTPase atlastin (ATL). Here, we have determined crystal structures of the cytosolic segment of human ATL1, which give insight into the mechanism of membrane fusion. The structures reveal a GTPase domain and athree-helix bundle, connected by a linker region. One structure corresponds to a prefusion state, in which ATL molecules in apposing membranes interact through their GTPase domains to form a dimer with the nucleotides bound at the interface. The other structure corresponds to a postfusion state generated after GTP hydrolysis and phosphate release. Compared with the prefusion structure, the three-helix bundles of the two ATL molecules undergo a major conformational change relative to the GTPase domains, which could pull the membranes together. The proposed fusion mechanism is supported by biochemical experiments and fusion assays with wild-type and mutant full-length Drosophila ATL. These experiments also show that membrane fusion is facilitated by the C-terminal cytosolic tails following the two transmembrane segments. Finally, our results show that mutations in ATL1 causing hereditary spastic paraplegia compromise homotypic ER fusion.

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Available from: Xuewu Sui, Apr 01, 2015
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    • "Atlastin molecules on the two opposed membranes have been proposed to transiently dimerize to attract the two membranes to each other (Bian et al., 2011;Byrnes and Sondermann, 2011;MorinLeisk et al., 2011;Moss et al., 2011;Lin et al., 2012;Byrnes et al., 2013). Closely attracted lipid bilayers are supposed to be destabilized by an amphipathic helical domain at the atlastin C terminus to facilitate membrane fusion (Bian et al., 2011;Liu et al., 2012;Faust et al., 2015). Knockdown of atlastins leads to fragmentation of the ER and unbranched ER tubules, while overexpression of atlastins enhances ER membrane fusion, which enlarges the ER profiles (Hu et al., 2009;Orso et al., 2009). "
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    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 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 cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in-vitro assay system of ER tubule formation with A. thaliana 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, serine/threonine-kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple serine and threonine residues in the C-terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the serine cluster was deleted or when the serine residues were replaced with alanine 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 have 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.
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    • "Atlastin molecules in different ER tubules form homodimers in trans in a GTP-dependent manner, thereby bringing these two membranes into close apposition (Orso et al., 2009). Upon GTP hydrolysis and Pi release, the cytosolic domain (CD) of the atlastin homodimers undergoes a dramatic conformational change, pulling the apposed membranes into close proximity and inducing membrane fusion (Bian et al., 2011; Byrnes and Sondermann, 2011). Second, ER-associated SNARE proteins are involved in homotypic ER fusion (Patel et al., 1998; Anwar et al., 2012). "
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    ABSTRACT: Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion. © 2015 Lee et al.
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    • "In S. cerevisiae, this fusion process is mediated by Sey1 via the formation of a homotypic dimer across opposite membranes in the ER. This dimerization is predicted to induce the GTPase activity of Sey1 and results in a protein conformation change that compels fusion of the two lipid bilayers (Hu et al., 2009; Orso et al., 2009; Bian et al., 2011; Byrnes and Sondermann, 2011; Anwar et al., 2012). Conversely, we previously identified Lnp1 as a regulator of ER tubule structure that seemingly acts as a counterbalance to Sey1 function. "
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    ABSTRACT: The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system yet harbor distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles including Rtn1, Yop1, Pom33, and Per33. Interestingly, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc-finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions. © 2015 by The American Society for Cell Biology.
    Preview · Article · Jun 2015 · Molecular biology of the cell
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