Cis and trans interactions between atlastin molecules during membrane fusion

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.

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Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission

The Journal of Cell Biology ◽

10.1083/jcb.200509095 ◽

2005 ◽

Vol 171 (6) ◽

pp. 919-924 ◽

Cited By ~ 138

Author(s):

Anna Bielli ◽

Charles J. Haney ◽

Gavin Gabreski ◽

Simon C. Watkins ◽

Sergei I. Bannykh ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Binding ◽

Guanosine Triphosphate ◽

Amphipathic Peptide ◽

Gtp Hydrolysis ◽

Membrane Deformation ◽

Gtp Binding ◽

Copii Vesicle ◽

Copii Vesicles ◽

And Control

The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271–277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.

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Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration

Molecular Biology of the Cell ◽

10.1091/mbc.e14-08-1284 ◽

2014 ◽

Vol 25 (24) ◽

pp. 3942-3953 ◽

Cited By ~ 22

Author(s):

Simran G. Saini ◽

Chuang Liu ◽

Peijun Zhang ◽

Tina H. Lee

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Potential Role ◽

Membrane Anchor ◽

Gtp Hydrolysis ◽

Nucleotide Hydrolysis ◽

Membrane Tethering ◽

In Trans ◽

Conformational Coupling

The membrane-anchored atlastin GTPase couples nucleotide hydrolysis to the catalysis of homotypic membrane fusion to form a branched endoplasmic reticulum network. Trans dimerization between atlastins anchored in opposing membranes, accompanied by a cross-over conformational change, is thought to draw the membranes together for fusion. Previous studies on the conformational coupling of atlastin to its GTP hydrolysis cycle have been carried out largely on atlastins lacking a membrane anchor. Consequently, whether fusion involves a discrete tethering step and, if so, the potential role of GTP hydrolysis and cross-over in tethering remain unknown. In this study, we used membrane-anchored atlastins in assays that separate tethering from fusion to dissect the requirements for each. We found that tethering depended on GTP hydrolysis, but, unlike fusion, it did not depend on cross-over. Thus GTP hydrolysis initiates stable head-domain contact in trans to tether opposing membranes, whereas cross-over formation plays a more pivotal role in powering the lipid rearrangements for fusion.

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Structures of the yeast dynamin-like GTPase Sey1p provide insight into homotypic ER fusion

The Journal of Cell Biology ◽

10.1083/jcb.201502078 ◽

2015 ◽

Vol 210 (6) ◽

pp. 961-972 ◽

Cited By ~ 31

Author(s):

Liming Yan ◽

Sha Sun ◽

Wei Wang ◽

Juanming Shi ◽

Xiaoyu Hu ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Candida Albicans ◽

Membrane Fusion ◽

Mammalian Cells ◽

Gtpase Activity ◽

Gtp Hydrolysis ◽

Helical Bundle ◽

The Common ◽

Insight Into ◽

Common Function

Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF4− but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p.

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GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion

The Journal of Cell Biology ◽

10.1083/jcb.201805039 ◽

2018 ◽

Vol 217 (12) ◽

pp. 4184-4198 ◽

Cited By ~ 4

Author(s):

James Winsor ◽

Ursula Machi ◽

Qixiu Han ◽

David D. Hackney ◽

Tina H. Lee

Keyword(s):

Membrane Fusion ◽

Conformational Change ◽

Initial Rate ◽

Fusion Activity ◽

Gtp Hydrolysis ◽

Nucleotide Hydrolysis ◽

Conformational Rearrangement ◽

Gtp Binding ◽

Over Time

Membrane fusion of the ER is catalyzed when atlastin GTPases anchored in opposing membranes dimerize and undergo a crossed over conformational rearrangement that draws the bilayers together. Previous studies have suggested that GTP hydrolysis triggers crossover dimerization, thus directly driving fusion. In this study, we make the surprising observations that WT atlastin undergoes crossover dimerization before hydrolyzing GTP and that nucleotide hydrolysis and Pi release coincide more closely with dimer disassembly. These findings suggest that GTP binding, rather than its hydrolysis, triggers crossover dimerization for fusion. In support, a new hydrolysis-deficient atlastin variant undergoes rapid GTP-dependent crossover dimerization and catalyzes fusion at an initial rate similar to WT atlastin. However, the variant cannot sustain fusion activity over time, implying a defect in subunit recycling. We suggest that GTP binding induces an atlastin conformational change that favors crossover dimerization for fusion and that the input of energy from nucleotide hydrolysis promotes complex disassembly for subunit recycling.

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Stimulation of glycosylphosphatidylinositol biosynthesis in mammaliancell-free systems by GTP hydrolysis: evidence for the involvement of membrane fusion

Biochemical Journal ◽

10.1042/bj3410577 ◽

1999 ◽

Vol 341 (3) ◽

pp. 577-584 ◽

Cited By ~ 4

Author(s):

Victoria L. STEVENS ◽

Hui ZHANG ◽

Eva Szucs KRISTYANNE

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Second Step ◽

Wild Type ◽

Gtp Hydrolysis ◽

Class A ◽

Stimulation Of

The second step in glycosylphosphatidylinositol (GPI) biosynthesis, the deacetylation of GlcNAc-phosphatidylinositol (GlcNAc-PI), has been shown to be stimulated by GTP hydrolysis [Stevens (1993) J. Biol. Chem. 268, 9718-9724]. We have now developed a system to study this regulation that uses microsomes from cells defective in the first step in GPI biosynthesis (class A, C and H lymphoma mutants) and the second reaction in the pathway (G9PLAP.85). With this mixed-microsome system, the deacetylation of GlcNAc-PI was almost completely dependent on GTP hydrolysis. Because GlcNAc-PI synthesized by the G9PLAP.85 microsomes cannot readily move to the first-step-mutant microsomes to be deacetylated, this result indicated that the role of GTP was to facilitate the ‘apparent’ transfer of this substrate between membrane vesicles. The microsomes could be stably preactivated by pretreatment with GTP before GPI biosynthesis was initiated, indicating that fusion was the most likely mechanism for this regulation. GlcNAc-PI deacetylation could also be stably preactivated in EL4 microsomes, suggesting that fusion also occurred in wild-type membranes. Some differential localization of the GlcNAc-PI synthetic and deacetylation activities with the endoplasmic reticulum was found. Therefore fusion seems to stimulate GPI biosynthesis in mammalian microsomes by bringing together the first two enzymes in the pathway in the same membrane vesicle.

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GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor.

The Journal of Cell Biology ◽

10.1083/jcb.123.4.799 ◽

1993 ◽

Vol 123 (4) ◽

pp. 799-807 ◽

Cited By ~ 42

Author(s):

T Connolly ◽

R Gilmore

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Signal Recognition Particle ◽

Hydrolysis Rate ◽

Endoplasmic Reticulum Membrane ◽

Signal Recognition ◽

Gtp Hydrolysis ◽

Receptor Complexes ◽

Gtp Binding ◽

Hydrolysis Activity

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP-dependent reaction during protein translocation is the SRP receptor-mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.

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