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

ABSTRACT 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 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.
    The Journal of Cell Biology 07/2015; 210(19). DOI:10.1083/jcb.201501043 · 9.83 Impact Factor
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    • "For instance , the side chain of Y196, a residue within the globular GTPase head, contacts a residue outside the head specifically in the crossover dimer configuration (Bian et al., 2011; Byrnes and Sondermann, 2011). The residue that it contacts, P342, resides within the linker connecting the head to the 3HB and constitutes the pivot point of 3HB rotation (Bian et al., 2011; Byrnes and Sondermann, 2011). On R214C, S233R, and S234Y (R239C, H258R, and S259Y in atlastin1), were clearly competent for fusion catalysis. "
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    ABSTRACT: At least 38 distinct missense mutations in the neuronal atlastin1/SPG3A GTPase are implicated in an autosomal dominant form of hereditary spastic paraplegia (HSP), a motor-neurological disorder manifested by lower limb weakness and spasticity and length dependent axonopathy of corticospinal motor neurons. Because the atlastin GTPase is sufficient to catalyze membrane fusion and required to form the ER network, at least in non-neuronal cells, it is logically assumed that defects in ER membrane morphogenesis due to impaired fusion activity are the primary drivers of SPG3A-associated HSP. Here we analyzed a subset of established atlastin1/SPG3A disease variants using cell-based assays for atlastin-mediated ER network formation and biochemical assays for atlastin-catalyzed GTP hydrolysis, dimer formation and membrane fusion. As anticipated, some variants exhibited clear deficits. Surprisingly however, at least two disease variants, one of which represents that most frequently identified in SPG3A HSP patients, displayed wild type levels of activity in all assays. The same variants were also capable of coredistributing ER-localized REEP1, a recently identified function of atlastins that requires its catalytic activity. Altogether, these findings indicate that a deficit in the membrane fusion activity of atlastin1 may be a key contributor, but not required, for HSP causation. © 2015 by The American Society for Cell Biology.
    Molecular Biology of the Cell 03/2015; 26(9). DOI:10.1091/mbc.E14-10-1447 · 4.47 Impact Factor
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    • "GADs are involved in numerous fundamental cellular functions . Important examples are membrane fusion and fission factors such as atlastin (Bian et al., 2011), dynamin (Chappie et al., 2010; Ford et al., 2011), and septin (Oh and Bi, 2011); the tRNAmodifying protein MnmE (Meyer et al., 2009), Xab1 (involved in DNA repair) (Gras et al., 2007; Nitta et al., 2000); and the signal recognition particle (SRP) and its receptor (SR) (Focia et al., 2004). The latter belong to the signal recognition GTPases and MinD and BioD (SIMIBI) superclass (Leipe et al., 2002). "
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    ABSTRACT: GTPases are molecular switches that control numerous crucial cellular processes. Unlike bona fide GTPases, which are regulated by intramolecular structural transitions, the less well studied GAD-GTPases are activated by nucleotide-dependent dimerization. A member of this family is the translocase of the outer envelope membrane of chloroplast Toc34 involved in regulation of preprotein import. The GTPase cycle of Toc34 is considered a major circuit of translocation regulation. Contrary to expectations, previous studies yielded only marginal structural changes of dimeric Toc34 in response to different nucleotide loads. Referencing PELDOR and FRET single-molecule and bulk experiments, we describe a nucleotide-dependent transition of the dimer flexibility from a tight GDP- to a flexible GTP-loaded state. Substrate binding induces an opening of the GDP-loaded dimer. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which likely represents a general regulatory mode for dimerizing GTPases.
    Structure 03/2014; 22(4). DOI:10.1016/j.str.2014.02.004 · 5.62 Impact Factor
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