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

Saini, Simran G.; Liu, Chuang; Zhang, Peijun; Lee, Tina H.

doi:10.1091/mbc.e14-08-1284

Cited by 32 publications

(87 citation statements)

References 21 publications

(117 reference statements)

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“…In addition, similar tightly "zippered" opposing membranes have been reported for fusionprimed membranes tethered by the atlastin GTP-driven fusion machinery (55). Moreover, it appears that during yeast vacuolar fusion, large extended membrane contacts form, though resolution of bilayers was not attained in that case (56).…”

Section: Discussionsupporting

confidence: 56%

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

Gui

Ebner

Mileant

et al. 2016

J Virol

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Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved. IMPORTANCEEnveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.

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Section: Discussionsupporting

confidence: 56%

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

Gui

Ebner

Mileant

et al. 2016

J Virol

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“…Rather, the rapid association of monomers in the GTP-bound state suggests that GTP hydrolysis most likely occurs after dimerization. It also has been suggested that tethering does not require the crossover configuration characteristic of crystal forms 1 and 3 and therefore would not involve a strong interaction among the 3HBs (25). This was based on a double mutant of ATL (P319G K320E), which was defective in generating the crossover configuration but was reported to have undiminished GTPase and tethering activity (25).…”

Section: Discussionmentioning

confidence: 99%

“…It also has been suggested that tethering does not require the crossover configuration characteristic of crystal forms 1 and 3 and therefore would not involve a strong interaction among the 3HBs (25). This was based on a double mutant of ATL (P319G K320E), which was defective in generating the crossover configuration but was reported to have undiminished GTPase and tethering activity (25). Although we agree that the mutant has GTPase activity similar to that of WT ATL, we found that it is significantly less active in tethering (SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, the data of Saini et al also point in this direction (compare figs 4 and 6F in ref. 25). Thus, it is difficult to make a strong point on the basis of this mutant.…”

Section: Discussionmentioning

confidence: 99%

“…On the addition of EDTA to the GTP-containing reaction, the vesicle size dropped to the original level, consistent with the absence of any fusion. While this manuscript was in preparation, another group also found that tethering requires GTP hydrolysis and can be separated from fusion (25).…”

Section: Gtp Hydrolysismentioning

confidence: 96%

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Cis and trans interactions between atlastin molecules during membrane fusion

Liu

Bian

Romano

et al. 2015

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

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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In VivoAnalysis of Membrane Fusion

Palfreyman

Jørgensen

2015

Encyclopedia of Life Sciences

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Membranes provide a barrier that allows chemical reactions to be isolated from the environment. The plasma membrane, for example, delineates self from nonself, and thus must have played an essential role in the evolution of life. Yet under numerous circumstances it is equally important that membranes be breached. Numerous forces oppose the spontaneous fusion of membranes; thus, specialized proteins have evolved to fuse membranes. The most well-understood fusion proteins are the viral fusion proteins and the SNARE proteins used in the secretory pathway. In addition, recent discoveries have lead to models for the fusion of organelles such as mitochondria and peroxisomes, as well as for cell-cell fusion. Despite the diverse structures of fusion proteins, it is possible that they function to drive membranes through a series of common lipid intermediates. Here we review the mechanisms of fusion for biological membranes, and highlight the similarities and differences in these processes.

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

Cited by 32 publications

References 21 publications

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

Cis and trans interactions between atlastin molecules during membrane fusion

In VivoAnalysis of Membrane Fusion

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