Membrane fusion by the GTPase atlastin requires a conserved C-terminal cytoplasmic tail and dimerization through the middle domain

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2011; 108(27):11133-8. DOI: 10.1073/pnas.1105056108
Source: PubMed


The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization.

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Available from: James A McNew, Oct 03, 2015
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    • "Unlabeled liposomes consisted of palmitoyloleoylphosphatidylcholine (POPC):dioleoyl phosphatidylserine (DOPS; 85:15), and labeled liposomes had POPC:DOPS:1,2-di- palmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3- benzoxadiazol-4-yl (DPPE-NBD):rhodamine-DPPE (82:15:1.5:1.5). Drosophila atlastin in 0.1% Anapoe-X 100 was reconstituted into preformed 100-nm liposomes as previously described (Moss et al., 2011). In brief, reconstitutions were carried out at a protein-to-lipid "
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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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    • "By a similar mechanism, the same proteins are also involved in generating the highly curved edges of ER sheets (Shibata et al. 2010). Two additional classes of proteins act in concert with RTNs and REEPs during ER morphogenesis: atlastins, large GTPases that mediate homotypic fusion of ER tubules, and spastin, an ATPase that separates microtubules, particularly in relation to membrane modeling events (Errico et al. 2002; Orlacchio et al. 2004; Connell et al. 2009; Hu et al. 2009; Bian et al. 2011; Morin-Leisk et al. 2011; Moss et al. 2011; Liu et al. 2012; Lumb et al. 2012). In line with this, the expression of an ATPase-defective spastin results in extensive tubulation of the ER, highlighting its role as a microtubule-severing protein during ER shaping (Connell et al. 2009). "
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    ABSTRACT: Reticulons (RTNs) are a group of membrane-associated proteins mainly responsible for shaping the tubular endoplasmic reticulum network, membrane trafficking, inhibition of axonal growth, and apoptosis. These proteins share a common sequence feature, the reticulon homology domain, which consists of paired hydrophobic stretches that are believed to induce membrane curvature by acting as a wedge in bilayer membranes. RTNs are ubiquitously expressed in all tissues, but each RTN member exhibits a unique expression pattern that prefers certain tissues or even cell types. Recently, accumulated evidence has suggested additional and unexpected roles for RTNs, including those on DNA binding, autophagy, and several inflammatory-related functions. These manifold actions of RTNs account for their ever-growing recognition of their involvement in neurodegenerative diseases like Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, as well as hereditary spastic paraplegia. This review summarizes the latest discoveries on RTNs in human pathophysiology, and the engagement of these in neurodegeneration, along with the implications of these findings for a better understanding of the molecular events triggered by RTNs and their potential exploitation as next-generation therapeutics.
    Neuromolecular medicine 11/2013; 16(1). DOI:10.1007/s12017-013-8271-9 · 3.68 Impact Factor
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    • "However, the structures depict only GDP-bound states, hence, the exact catalytic mechanism for atlastin and the concurrent conformational switching upon GTP hydrolysis remained unknown. It has also been established that the middle domain is required for dimerization and GTPase activity (Morin-Leisk et al, 2011; Moss et al, 2011; Pendin et al, 2011), the reason for which is not obvious from the initial models. In addition, a recent report demonstrated, using single-particle electron microscopy, that atlastin-2 bound to the GTP-mimic GppNHp adopts both the presumed pre-and post-fusion conformations, the latter of which depends on an intramolecular salt bridge between the middle domain and the adjacent linker (Morin-Leisk et al, 2011). "
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    ABSTRACT: Atlastin, a member of the dynamin superfamily, is known to catalyse homotypic membrane fusion in the smooth endoplasmic reticulum (ER). Recent studies of atlastin have elucidated key features about its structure and function; however, several mechanistic details, including the catalytic mechanism and GTP hydrolysis-driven conformational changes, are yet to be determined. Here, we present the crystal structures of atlastin-1 bound to GDP·AlF(4)(-) and GppNHp, uncovering an intramolecular arginine finger that stimulates GTP hydrolysis when correctly oriented through rearrangements within the G domain. Utilizing Förster Resonance Energy Transfer, we describe nucleotide binding and hydrolysis-driven conformational changes in atlastin and their sequence. Furthermore, we discovered a nucleotide exchange mechanism that is intrinsic to atlastin's N-terminal domains. Our results indicate that the cytoplasmic domain of atlastin acts as a tether and homotypic interactions are timed by GTP binding and hydrolysis. Perturbation of these mechanisms may be implicated in a group of atlastin-associated hereditary neurodegenerative diseases.
    The EMBO Journal 01/2013; 32(3). DOI:10.1038/emboj.2012.353 · 10.43 Impact Factor
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