Comparison of human and Drosophila atlastin GTPases

Wu, Fuyun; Hu, Xiaoyu; Bian, Xin; Liu, Xinqi; Hu, Junjie

doi:10.1007/s13238-014-0118-0

Cited by 26 publications

(44 citation statements)

References 20 publications

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“…Whereas Drosophila atlastin is an efficient fusogen in vitro (5,(31)(32)(33)(34)(35), the human atlastin proteins have been resistant to in vitro fusion analysis (36). We have confirmed that human atlastin-1 does not significantly promote membrane fusion in vitro (Fig.…”

Section: Biochemical Analysis Of Human Atlastin-1 and Human-drosophilsupporting

confidence: 63%

The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer

Betancourt-Solis

Desai²,

McNew

2018

Journal of Biological Chemistry

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The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. Here, we examined several Drosophila-human atlastin chimeras to identify functional domains of human atlastin-1 in vitro. Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro. We also determined that co-reconstitution of atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, we found that both human and Drosophila atlastin hydrophobic membrane anchors do not span the membrane, but rather form two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.

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Section: Biochemical Analysis Of Human Atlastin-1 and Human-drosophilsupporting

confidence: 63%

The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer

Betancourt-Solis

Desai²,

McNew

2018

Journal of Biological Chemistry

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“…The protein is bound to GDP despite being crystalized in the presence of GTPγS and Mg 2+ . Globally, the hATL3 structure resembles earlier structures of hATL1 and dmATL bound to GDP•Mg 2+ where the G and middle domains are engaged and individual protomers are forming a weak dimer interface between G domains (interface surface area = 780 Å 2 , Δ i G = −5.9 kcal/mol (Krissinel and Henrick, 2007)) (Figure 6A) (Bian et al, 2011; Byrnes and Sondermann, 2011; Wu et al, 2015). One of the hallmarks of this functional state, a central helix, α4, that is bent as a result of the G-middle domain docking, is also preserved in hATL3 (Figure 6B).…”

Section: Resultsmentioning

confidence: 60%

“…The hATL3 structure revealed a novel active site organization: whereas hATL1 (PDB 3Q5E), dmATL (PDB 3X1D), and Sey1 (PDB 5CA8) exhibit the same binding configuration for GDP•Mg 2+ (Byrnes and Sondermann, 2011; Wu et al, 2015; Yan et al, 2015), the canonical Mg 2+ was displaced by the guanidinium group of an intramolecular arginine residue at position 109 (R 109 ) in the hATL3 structure (Figures 6C and 6D), an active site residue that is distinct from the catalytic arginine finger (R 70 in hATL3, R 77 in hATL1). The conformation of R 109 is further stabilized through a salt-bridge formed with an aspartate residue at position 142, one of the main Mg 2+ -coordinating residues in ATLs (Figure 6C) (Bian et al, 2011; Byrnes and Sondermann, 2011).…”

Section: Resultsmentioning

confidence: 99%

“…Notably, the core fragment is an effective GTPase-dependent inhibitor of liposome fusion and ER formation in reconstituted Xenopus egg extracts (Moss et al, 2011; Wang et al, 2013; Wang et al, 2016), which argues that this core fragment is able to dimerize with full-length ATL during the latter’s native reaction cycle. The catalytic core also has comparable GTPase activity to the full-length protein and is sufficient for nucleotide-dependent vesicle tethering when immobilized to Ni 2+ -nitrilotriacetic acid (NTA) lipids via a C-terminal hexa-histidine tag (Liu et al, 2015; Moss et al, 2011; Wu et al 2015). Hence, the catalytic core has proven to be a valuable proxy for understanding the full-length protein in regard to GTPase-driven steps of the reaction cycle.…”

Section: Introductionmentioning

confidence: 99%

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Timing and Reset Mechanism of GTP Hydrolysis-Driven Conformational Changes of Atlastin

et al. 2017

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Summary The endoplasmic reticulum (ER) forms a branched, dynamic membrane tubule network that is vital for cellular function. Branching arises from membrane fusion facilitated by the GTPase atlastin (ATL). Many metazoan genomes encode for three ATL isoforms that appear to fulfill partially redundant function despite differences in their intrinsic GTPase activity and localization within the ER; however, the underlying mechanistic differences between the isoforms are poorly understood. Here, we identify discrete temporal steps in the catalytic cycle for the two most dissimilar isoforms, ATL1 and ATL3, revealing an overall conserved progression of molecular events from nucleotide binding and hydrolysis, to ATL dimerization and phosphate release. A crystal structure of ATL3 suggests a mechanism for the displacement of the catalytic Mg2+ ion following GTP hydrolysis. Together, the data extend the mechanistic framework for how GTP hydrolysis drives conformational changes in ATL and how the cycle is reset for subsequent rounds of catalysis.

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“…This structure resembles the postfusion state rather than the expected prefusion state; the two 3HBs are again parallel, and in fact are even closer to one another. Finally, a structure of GDP-bound Drosophila ATL showed a prefusion-like conformation (20).…”

Section: Significancementioning

confidence: 97%

Cis and trans interactions between atlastin molecules during membrane fusion

Liu

Bian

Romano

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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115

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Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions. T he endoplasmic reticulum (ER) consists of tubules and sheets connected into a characteristic network by membrane fusion (1-3). In contrast to heterotypic fusion between viral and cellular membranes or between intracellular transport vesicles and target membranes, the fusing ER membranes are identical (i.e., homotypic fusion). The mechanism of heterotypic fusion has been studied extensively, leading to the concept that a conformational change of a viral fusion protein or the assembly of SNARE proteins pulls two opposing membranes together so that they can fuse (4-7). Recently, some insight has been obtained into homotypic ER fusion as well. This process is mediated by the atlastins (ATLs) in metazoans and by Sey1p/ROOT HAIR DEFECTIVE3 (RHD3)-related proteins in yeast and plants (8,9).The ATLs and Sey1p/RHD3 are membrane-bound GTPases that belong to the dynamin family (reviewed in refs. 10, 11). They contain cytosolic N-terminal GTPase (G) and helical bundle domains, followed by two closely spaced transmembrane (TM) segments and a cytosolic C-terminal tail (12)(13)(14). A role for the GTPases in ER fusion is suggested by the observation that their depletion or mutational inactivation leads to long, nonbranched tubules or fragmented ER (8, 9). Nonbranched ER tubules are also observed on expression of dominant-negative ATL mutants (8,12). In addition, the fusion of ER vesicles in Xenopus laevis egg extracts is prevented by ATL antibodies or a cytosolic fragment of ATL (8,15), and the fusion of the ER in mating yeast cells is delayed on SEY1 deletion (16). Most convincingly, proteoliposomes containing purified ATL, Sey1p, or RHD3 undergo GTP-dependent fusion in vitro (9,13,16,17). Defects in ATL-mediated ER fusion can cause hereditary spastic paraplegia, a neurodegenerativ...

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Comparison of human and Drosophila atlastin GTPases

Cited by 26 publications

References 20 publications

The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer

The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer

Timing and Reset Mechanism of GTP Hydrolysis-Driven Conformational Changes of Atlastin

Cis and trans interactions between atlastin molecules during membrane fusion

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