Journal articles on the topic 'Protein Mediated Membrane Fusion'
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1
Stegmann, T., R. W. Doms, and A. Helenius. "Protein-Mediated Membrane Fusion." Annual Review of Biophysics and Biophysical Chemistry 18, no. 1 (June 1989): 187–211. http://dx.doi.org/10.1146/annurev.bb.18.060189.001155.
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Benhaim, Mark A., and Kelly K. Lee. "New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes." Viruses 12, no. 4 (April 8, 2020): 413. http://dx.doi.org/10.3390/v12040413.
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Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.
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Boonstra, Sander, Jelle S. Blijleven, Wouter H. Roos, Patrick R. Onck, Erik van der Giessen, and Antoine M. van Oijen. "Hemagglutinin-Mediated Membrane Fusion: A Biophysical Perspective." Annual Review of Biophysics 47, no. 1 (May 20, 2018): 153–73. http://dx.doi.org/10.1146/annurev-biophys-070317-033018.
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Influenza hemagglutinin (HA) is a viral membrane protein responsible for the initial steps of the entry of influenza virus into the host cell. It mediates binding of the virus particle to the host-cell membrane and catalyzes fusion of the viral membrane with that of the host. HA is therefore a major target in the development of antiviral strategies. The fusion of two membranes involves high activation barriers and proceeds through several intermediate states. Here, we provide a biophysical description of the membrane fusion process, relating its kinetic and thermodynamic properties to the large conformational changes taking place in HA and placing these in the context of multiple HA proteins working together to mediate fusion. Furthermore, we highlight the role of novel single-particle experiments and computational approaches in understanding the fusion process and their complementarity with other biophysical approaches.
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Kumar, Pawan, Samit Guha, and Ulf Diederichsen. "SNARE protein analog-mediated membrane fusion." Journal of Peptide Science 21, no. 8 (April 7, 2015): 621–29. http://dx.doi.org/10.1002/psc.2773.
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Stiasny, Karin, and Franz X. Heinz. "Flavivirus membrane fusion." Journal of General Virology 87, no. 10 (October 1, 2006): 2755–66. http://dx.doi.org/10.1099/vir.0.82210-0.
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Flavivirus membrane fusion is mediated by a class II viral fusion protein, the major envelope protein E, and the fusion process is extremely fast and efficient. Understanding of the underlying mechanisms has been advanced significantly by the determination of E protein structures in their pre- and post-fusion conformations and by the elucidation of the quarternary organization of E proteins in the viral envelope. In this review, these structural data are discussed in the context of functional and biochemical analyses of the flavivirus fusion mechanism and its characteristics are compared with those of other class II- and class I-driven fusion processes.
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Fu, Jiawen, Lin Zhao, Juan Yang, Heming Chen, Shinuo Cao, and Honglin Jia. "An unconventional SNARE complex mediates exocytosis at the plasma membrane and vesicular fusion at the apical annuli in Toxoplasma gondii." PLOS Pathogens 19, no. 3 (March 27, 2023): e1011288. http://dx.doi.org/10.1371/journal.ppat.1011288.
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Exocytosis is a key active process in cells by which proteins are released in bulk via the fusion of exocytic vesicles with the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-mediated vesicle fusion with the plasma membrane is essential in most exocytotic pathways. In mammalian cells, the vesicular fusion step of exocytosis is normally mediated by Syntaxin-1 (Stx1) and SNAP25 family proteins (SNAP25 and SNAP23). However, in Toxoplasma gondii, a model organism of Apicomplexa, the only SNAP25 family protein, with a SNAP29-like molecular structure, is involved in vesicular fusion at the apicoplast. Here, we reveal that an unconventional SNARE complex comprising TgStx1, TgStx20, and TgStx21 mediates vesicular fusion at the plasma membrane. This complex is essential for the exocytosis of surface proteins and vesicular fusion at the apical annuli in T. gondii.
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Taylor, Gwen M., and David Avram Sanders. "The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion." Molecular Biology of the Cell 10, no. 9 (September 1999): 2803–15. http://dx.doi.org/10.1091/mbc.10.9.2803.
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The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.
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Johnson, Colin P., and Edwin R. Chapman. "Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion." Journal of Cell Biology 191, no. 1 (October 4, 2010): 187–97. http://dx.doi.org/10.1083/jcb.201002089.
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Otoferlin is a large multi–C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13–25 µM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding–deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein’s function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated membrane fusion reactions.
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Kingsley, David H., Ali Behbahani, Afshin Rashtian, Gary W. Blissard, and Joshua Zimmerberg. "A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion." Molecular Biology of the Cell 10, no. 12 (December 1999): 4191–200. http://dx.doi.org/10.1091/mbc.10.12.4191.
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Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.
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Grothe, Tobias, Julia Nowak, Reinhard Jahn, and Peter Jomo Walla. "Selected tools to visualize membrane interactions." European Biophysics Journal 50, no. 2 (March 2021): 211–22. http://dx.doi.org/10.1007/s00249-021-01516-6.
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AbstractIn the past decade, we developed various fluorescence-based methods for monitoring membrane fusion, membrane docking, distances between membranes, and membrane curvature. These tools were mainly developed using liposomes as model systems, which allows for the dissection of specific interactions mediated by, for example, fusion proteins. Here, we provide an overview of these methods, including two-photon fluorescence cross-correlation spectroscopy and intramembrane Förster energy transfer, with asymmetric labelling of inner and outer membrane leaflets and the calibrated use of transmembrane energy transfer to determine membrane distances below 10 nm. We discuss their application range and their limitations using examples from our work on protein-mediated vesicle docking and fusion.
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Diao, Jiajie, Yuji Ishitsuka, and Woo-Ri Bae. "Single-molecule FRET study of SNARE-mediated membrane fusion." Bioscience Reports 31, no. 6 (September 15, 2011): 457–63. http://dx.doi.org/10.1042/bsr20110011.
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Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.
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Weber-Boyvat, Marion, Hongxia Zhao, Nina Aro, Qiang Yuan, Konstantin Chernov, Johan Peränen, Pekka Lappalainen, and Jussi Jäntti. "A conserved regulatory mode in exocytic membrane fusion revealed by Mso1p membrane interactions." Molecular Biology of the Cell 24, no. 3 (February 2013): 331–41. http://dx.doi.org/10.1091/mbc.e12-05-0415.
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Sec1/Munc18 family proteins are important components of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex–mediated membrane fusion processes. However, the molecular interactions and the mechanisms involved in Sec1p/Munc18 control and SNARE complex assembly are not well understood. We provide evidence that Mso1p, a Sec1p- and Sec4p-binding protein, interacts with membranes to regulate membrane fusion. We identify two membrane-binding sites on Mso1p. The N-terminal region inserts into the lipid bilayer and appears to interact with the plasma membrane, whereas the C-terminal region of the protein binds phospholipids mainly through electrostatic interactions and may associate with secretory vesicles. The Mso1p membrane interactions are essential for correct subcellular localization of Mso1p–Sec1p complexes and for membrane fusion in Saccharomyces cerevisiae. These characteristics are conserved in the phosphotyrosine-binding (PTB) domain of β-amyloid precursor protein–binding Mint1, the mammalian homologue of Mso1p. Both Mint1 PTB domain and Mso1p induce vesicle aggregation/clustering in vitro, supporting a role in a membrane-associated process. The results identify Mso1p as a novel lipid-interacting protein in the SNARE complex assembly machinery. Furthermore, our data suggest that a general mode of interaction, consisting of a lipid-binding protein, a Rab family GTPase, and a Sec1/Munc18 family protein, is important in all SNARE-mediated membrane fusion events.
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Hughson, Frederick M. "Molecular mechanisms of protein-mediated membrane fusion." Current Opinion in Structural Biology 5, no. 4 (August 1995): 507–13. http://dx.doi.org/10.1016/0959-440x(95)80036-0.
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Wessels, Laura, and Keith Weninger. "Physical Aspects of Viral Membrane Fusion." Scientific World JOURNAL 9 (2009): 764–80. http://dx.doi.org/10.1100/tsw.2009.76.
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Enveloped viruses commonly employ membrane fusion during cell penetration in order to deliver their genetic material across the cell boundary. Large conformational changes in the proteins embedded in the viral membrane play a fundamental role in the membrane fusion process. Despite the tremendously wide variety of viruses that contain membranes, it appears that they all contain membrane fusion protein machinery with a remarkably conserved mechanism of action. Much of our current biochemical understanding of viral membrane fusion has been derived from high-resolution structural studies and solution-basedin vitroassays in which viruses fuse with liposomes or cells. Recently, single-particle experiments have been used to provide measurements of details not available in the bulk assays. Here we focus our discussion on the key dynamical aspects of fusion protein structure, along with some of the experimental and computational techniques presently being used to investigate viral-mediated membrane fusion.
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Liao, Maofu, and Margaret Kielian. "Functions of the Stem Region of the Semliki Forest Virus Fusion Protein during Virus Fusion and Assembly." Journal of Virology 80, no. 22 (September 13, 2006): 11362–69. http://dx.doi.org/10.1128/jvi.01679-06.
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ABSTRACT Membrane fusion of the alphaviruses is mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 dissociates from its heterodimer interaction with the E2 protein and forms a target membrane-inserted E1 homotrimer. The structure of the homotrimer is that of a trimeric hairpin in which E1 domain III and the stem region fold back toward the target membrane-inserted fusion peptide loop. The E1 stem region has a strictly conserved length and several highly conserved residues, suggesting the possibility of specific stem interactions along the trimer core and an important role in driving membrane fusion. Mutagenesis studies of the alphavirus Semliki Forest virus (SFV) here demonstrated that there was a strong requirement for the E1 stem in virus assembly and budding, probably reflecting its importance in lateral interactions of the envelope proteins. Surprisingly, however, neither the conserved length nor any specific residues of the stem were required for membrane fusion. Although the highest fusion activity was observed with wild-type E1, efficient fusion was mediated by stem mutants containing a variety of substitutions or deletions. A minimal stem length was required but could be conferred by a series of alanine residues. The lack of a specific stem sequence requirement during SFV fusion suggests that the interaction of domain III with the trimer core can provide sufficient driving force to mediate membrane merger.
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Stiasny, Karin, and Franz X. Heinz. "Effect of Membrane Curvature-Modifying Lipids on Membrane Fusion by Tick-Borne Encephalitis Virus." Journal of Virology 78, no. 16 (August 15, 2004): 8536–42. http://dx.doi.org/10.1128/jvi.78.16.8536-8542.2004.
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ABSTRACT Enveloped viruses enter cells by fusion of their own membrane with a cellular membrane. Incorporation of inverted-cone-shaped lipids such as lysophosphatidylcholine (LPC) into the outer leaflet of target membranes has been shown previously to impair fusion mediated by class I viral fusion proteins, e.g., the influenza virus hemagglutinin. It has been suggested that these results provide evidence for the stalk-pore model of fusion, which involves a hemifusion intermediate (stalk) with highly bent outer membrane leaflets. Here, we investigated the effect of inverted-cone-shaped LPCs and the cone-shaped oleic acid (OA) on the membrane fusion activity of a virus with a class II fusion protein, the flavivirus tick-borne encephalitis virus (TBEV). This study included an analysis of lipid mixing, as well as of the steps preceding or accompanying fusion, i.e., binding to the target membrane and lipid-induced conformational changes in the fusion protein E. We show that the presence of LPC in the outer leaflet of target liposomes strongly inhibited TBEV-mediated fusion, whereas OA caused a very slight enhancement, consistent with a fusion mechanism involving a lipid stalk. However, LPC also impaired the low-pH-induced binding of a soluble form of the E protein to liposomes and its conversion into a trimeric postfusion structure that requires membrane binding at low pH. Because inhibition is already observed before the lipid-mixing step, it cannot be determined whether impairment of stalk formation is a contributing factor in the inhibition of fusion by LPC. These data emphasize, however, the importance of the composition of the target membrane in its interactions with the fusion peptide that are crucial for the initiation of fusion.
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Gui, Long, Jamie L. Ebner, Alexander Mileant, James A. Williams, and Kelly K. Lee. "Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion." Journal of Virology 90, no. 15 (May 25, 2016): 6948–62. http://dx.doi.org/10.1128/jvi.00240-16.
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ABSTRACTProtein-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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McClain, Mark S., Ping Cao, and Timothy L. Cover. "Amino-Terminal Hydrophobic Region ofHelicobacter pylori Vacuolating Cytotoxin (VacA) Mediates Transmembrane Protein Dimerization." Infection and Immunity 69, no. 2 (February 1, 2001): 1181–84. http://dx.doi.org/10.1128/iai.69.2.1181-1184.2001.
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ABSTRACT Helicobacter pylori VacA is a secreted protein toxin that forms channels in lipid bilayers and induces multiple structural and functional alterations in eukaryotic cells. A unique hydrophobic segment at the amino terminus of VacA contains three tandem repeats of a GxxxG motif that is characteristic of transmembrane dimerization sequences. To examine functional properties of this region, we expressed and analyzed ToxR-VacA-maltose binding protein fusions using the TOXCAT system, which was recently developed by W. P. Russ and D. M. Engelman (Proc. Natl. Acad. Sci. USA 96:863–868, 1999) to study transmembrane helix-helix associations in a natural membrane environment. A wild-type VacA hydrophobic region mediated insertion of the fusion protein into the inner membrane of Escherichia coli and mediated protein dimerization. A fusion protein containing a mutant VacA hydrophobic region (in which glycine 14 of VacA was replaced by alanine) also inserted into the inner membrane but dimerized significantly less efficiently than the fusion protein containing the wild-type VacA sequence. Based on these results, we speculate that the wild-type VacA amino-terminal hydrophobic region contributes to oligomerization of the toxin within membranes of eukaryotic cells.
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Chanel-Vos, Chantal, and Margaret Kielian. "Second-Site Revertants of a Semliki Forest Virus Fusion-Block Mutation Reveal the Dynamics of a Class II Membrane Fusion Protein." Journal of Virology 80, no. 12 (June 15, 2006): 6115–22. http://dx.doi.org/10.1128/jvi.00167-06.
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ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced membrane fusion mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 inserts into target membranes via its hydrophobic fusion loop and refolds to form a stable E1 homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the fusion loop was previously shown to block SFV fusion and infection, although the mutant E1 protein still inserts into target membranes and forms a homotrimer. Here we report on second-site mutations in E1 that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the membrane-interacting tip, and within the groove where the E1 stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the fusion protein refolding reaction.
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Ou, Wu, and Jonathan Silver. "Stoichiometry of Murine Leukemia Virus Envelope Protein-Mediated Fusion and Its Neutralization." Journal of Virology 80, no. 24 (October 11, 2006): 11982–90. http://dx.doi.org/10.1128/jvi.01318-06.
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ABSTRACT Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.
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Ahn, Anna, Don L. Gibbons, and Margaret Kielian. "The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains." Journal of Virology 76, no. 7 (April 1, 2002): 3267–75. http://dx.doi.org/10.1128/jvi.76.7.3267-3275.2002.
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ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl β-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.
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Martens, Sascha. "Role of C2 domain proteins during synaptic vesicle exocytosis." Biochemical Society Transactions 38, no. 1 (January 19, 2010): 213–16. http://dx.doi.org/10.1042/bst0380213.
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Neurotransmitter release is mediated by the fusion of synaptic vesicles with the presynaptic plasma membrane. Fusion is triggered by a rise in the intracellular calcium concentration and is dependent on the neuronal SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complex. A plethora of molecules such as members of the MUNC13, MUNC18, complexin and synaptotagmin families act along with the SNARE complex to enable calcium-regulated synaptic vesicle exocytosis. The synaptotagmins are localized to synaptic vesicles by an N-terminal transmembrane domain and contain two cytoplasmic C2 domains. Members of the synaptotagmin family are thought to translate the rise in intracellular calcium concentration into synaptic vesicle fusion. The C2 domains of synaptotagmin-1 bind membranes in a calcium-dependent manner and in response induce a high degree of membrane curvature, which is required for its ability to trigger membrane fusion in vitro and in vivo. Furthermore, members of the soluble DOC2 (double-C2 domain) protein family have similar properties. Taken together, these results suggest that C2 domain proteins such as the synaptotagmins and DOC2s promote membrane fusion by the induction of membrane curvature in the vicinity of the SNARE complex. Given the widespread expression of C2 domain proteins in secretory cells, it is proposed that promotion of SNARE-dependent membrane fusion by the induction of membrane curvature is a widespread phenomenon.
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Duncan, Roy. "Fusogenic Reoviruses and Their Fusion-Associated Small Transmembrane (FAST) Proteins." Annual Review of Virology 6, no. 1 (September 29, 2019): 341–63. http://dx.doi.org/10.1146/annurev-virology-092818-015523.
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With no limiting membrane surrounding virions, nonenveloped viruses have no need for membrane fusion to gain access to intracellular replication compartments. Consequently, nonenveloped viruses do not encode membrane fusion proteins. The only exception to this dogma is the fusogenic reoviruses that encode fusion-associated small transmembrane (FAST) proteins that induce syncytium formation. FAST proteins are the smallest viral membrane fusion proteins and, unlike their enveloped virus counterparts, are nonstructural proteins that evolved specifically to induce cell-to-cell, not virus-cell, membrane fusion. This distinct evolutionary imperative is reflected in structural and functional features that distinguish this singular family of viral fusogens from all other protein fusogens. These rudimentary fusogens comprise specific combinations of different membrane effector motifs assembled into small, modular membrane fusogens. FAST proteins offer a minimalist model to better understand the ubiquitous process of protein-mediated membrane fusion and to reveal novel mechanisms of nonenveloped virus dissemination that contribute to virulence.
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Kobayashi, Mariko, Michael C. Bennett, Theodore Bercot, and Ila R. Singh. "Functional Analysis of Hepatitis C Virus Envelope Proteins, Using a Cell-Cell Fusion Assay." Journal of Virology 80, no. 4 (February 15, 2006): 1817–25. http://dx.doi.org/10.1128/jvi.80.4.1817-1825.2006.
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ABSTRACT Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.
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Jain, Surbhi, Lori W. McGinnes, and Trudy G. Morrison. "Overexpression of Thiol/Disulfide Isomerases Enhances Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein." Journal of Virology 82, no. 24 (October 1, 2008): 12039–48. http://dx.doi.org/10.1128/jvi.01406-08.
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ABSTRACT Newcastle disease virus (NDV) fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. We have previously shown that free thiols are present in cell surface-expressed NDV F protein and that blocking the production of free thiols by thiol-disulfide exchange inhibitors inhibited the membrane fusion mediated by F protein (J Virol. 81:2328-2339, 2007). Extending these observations, we evaluated the role of the overexpression of two disulfide bond isomerases, protein disulfide isomerase (PDI) and ERdj5, in cell-cell fusion mediated by NDV glycoproteins. The overexpression of these isomerases resulted in significantly increased membrane fusion, as measured by syncytium formation and content mixing. The overexpression of these isomerases enhanced the production of free thiols in F protein when expressed without hemagglutination-neuraminidase (HN) protein but decreased free thiols in F protein expressed with HN protein. By evaluating the binding of conformation-sensitive antibodies, we found that the overexpression of these isomerases favored a postfusion conformation of surface-expressed F protein in the presence of HN protein. These results suggest that isomerases belonging to the PDI family catalyze the production of free thiols in F protein, and free thiols in F protein facilitate membrane fusion mediated by F protein.
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Yang, Yiming, and Nandini Nagarajan Margam. "Structural Insights into Membrane Fusion Mediated by Convergent Small Fusogens." Cells 10, no. 1 (January 15, 2021): 160. http://dx.doi.org/10.3390/cells10010160.
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From lifeless viral particles to complex multicellular organisms, membrane fusion is inarguably the important fundamental biological phenomena. Sitting at the heart of membrane fusion are protein mediators known as fusogens. Despite the extensive functional and structural characterization of these proteins in recent years, scientists are still grappling with the fundamental mechanisms underlying membrane fusion. From an evolutionary perspective, fusogens follow divergent evolutionary principles in that they are functionally independent and do not share any sequence identity; however, they possess structural similarity, raising the possibility that membrane fusion is mediated by essential motifs ubiquitous to all. In this review, we particularly emphasize structural characteristics of small-molecular-weight fusogens in the hope of uncovering the most fundamental aspects mediating membrane–membrane interactions. By identifying and elucidating fusion-dependent functional domains, this review paves the way for future research exploring novel fusogens in health and disease.
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Wahlberg, J. M., and H. Garoff. "Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells." Journal of Cell Biology 116, no. 2 (January 15, 1992): 339–48. http://dx.doi.org/10.1083/jcb.116.2.339.
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The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells. Existing evidence suggests that the E1 protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the original E2E1 heterodimer is destabilized and the E1 proteins form new oligomers, presumably homooligomers, with altered E1 structure. This altered structure of E1 is specifically recognized by a monoclonal antibody which can also inhibit penetration of SFV into host cells as well as SFV-mediated cell-cell fusion, thus suggesting that the altered E1 structure is important for the membrane fusion. These results give further support for a membrane protein oligomerization-mediated control mechanism for the membrane fusion potential in alphaviruses.
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Yu, Haijia, Chong Shen, Yinghui Liu, Bridget L. Menasche, Yan Ouyang, Michael H. B. Stowell, and Jingshi Shen. "SNARE zippering requires activation by SNARE-like peptides in Sec1/Munc18 proteins." Proceedings of the National Academy of Sciences 115, no. 36 (August 20, 2018): E8421—E8429. http://dx.doi.org/10.1073/pnas.1802645115.
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Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE–SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE–SLP intermediate must form before SNAREs can drive efficient vesicle fusion.
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Liao, Maofu, Claudia Sánchez-San Martín, Aihua Zheng, and Margaret Kielian. "In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein." Journal of Virology 84, no. 11 (March 24, 2010): 5730–40. http://dx.doi.org/10.1128/jvi.00170-10.
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ABSTRACT The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.
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Yamamoto, Mizuki, Takeshi Ichinohe, Aya Watanabe, Ayako Kobayashi, Rui Zhang, Jiping Song, Yasushi Kawaguchi, Zene Matsuda, and Jun-ichiro Inoue. "The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion." Viruses 12, no. 12 (December 21, 2020): 1475. http://dx.doi.org/10.3390/v12121475.
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Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6–2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.
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Richard, Jean-Philippe, Evgenia Leikina, Ralf Langen, William Mike Henne, Margarita Popova, Tamas Balla, Harvey T. McMahon, Michael M. Kozlov, and Leonid V. Chernomordik. "Intracellular curvature-generating proteins in cell-to-cell fusion." Biochemical Journal 440, no. 2 (November 14, 2011): 185–93. http://dx.doi.org/10.1042/bj20111243.
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Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, in the present study we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature-generating proteins in cells by either expressing or microinjecting the ENTH (epsin N-terminal homology) domain of epsin or by expressing the GRAF1 (GTPase regulator associated with focal adhesion kinase 1) BAR (Bin/amphiphysin/Rvs) domain or the FCHo2 (FCH domain-only protein 2) F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature-generating protein, dynamin. Cell-membrane-permeant inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also report that syncytium formation is inhibited by reagents lowering the content and accessibility of PtdIns(4,5)P2, an important regulator of intracellular membrane remodelling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins.
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Salsman, Jayme, Deniz Top, Julie Boutilier, and Roy Duncan. "Extensive Syncytium Formation Mediated by the Reovirus FAST Proteins Triggers Apoptosis-Induced Membrane Instability." Journal of Virology 79, no. 13 (July 1, 2005): 8090–100. http://dx.doi.org/10.1128/jvi.79.13.8090-8100.2005.
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ABSTRACT The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by nonenveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively nonleaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ectodomain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic nonenveloped viruses, with nonleaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.
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Bruns, Caroline, J. Michael McCaffery, Amy J. Curwin, Juan M. Duran, and Vivek Malhotra. "Biogenesis of a novel compartment for autophagosome-mediated unconventional protein secretion." Journal of Cell Biology 195, no. 6 (December 5, 2011): 979–92. http://dx.doi.org/10.1083/jcb.201106098.
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The endoplasmic reticulum (ER)–Golgi-independent, unconventional secretion of Acb1 requires many different proteins. They include proteins necessary for the formation of autophagosomes, proteins necessary for the fusion of membranes with the endosomes, proteins of the multivesicular body pathway, and the cell surface target membrane SNARE Sso1, thereby raising the question of what achieves the connection between these diverse proteins and Acb1 secretion. In the present study, we now report that, upon starvation in Saccharomyces cerevisiae, Grh1 is collected into unique membrane structures near Sec13-containing ER exit sites. Phosphatidylinositol 3 phosphate, the ESCRT (endosomal sorting complex required for transport) protein Vps23, and the autophagy-related proteins Atg8 and Atg9 are recruited to these Grh1-containing membranes, which lack components of the Golgi apparatus and the endosomes, and which we call a novel compartment for unconventional protein secretion (CUPS). We describe the cellular proteins required for the biogenesis of CUPS, which we believe is the sorting station for Acb1’s release from the cells.
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Su, B., G. L. Waneck, R. A. Flavell, and A. L. Bothwell. "The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation." Journal of Cell Biology 112, no. 3 (February 1, 1991): 377–84. http://dx.doi.org/10.1083/jcb.112.3.377.
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Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.
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Arnold, Matthew Grant, Pratikshya Adhikari, Baobin Kang, and Hao Xu (徐昊). "Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering." Biochemical Journal 474, no. 19 (September 25, 2017): 3339–54. http://dx.doi.org/10.1042/bcj20170494.
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Sec1–Munc18 (SM) proteins co-operate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here, we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.
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Sato, Miyuki, Keiko Saegusa, Katsuya Sato, Taichi Hara, Akihiro Harada, and Ken Sato. "Caenorhabditis elegans SNAP-29 is required for organellar integrity of the endomembrane system and general exocytosis in intestinal epithelial cells." Molecular Biology of the Cell 22, no. 14 (July 15, 2011): 2579–87. http://dx.doi.org/10.1091/mbc.e11-04-0279.
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It is generally accepted that soluble N-ethylmaleimide–sensitive factor attachment protein receptors mediate the docking and fusion of transport intermediates with target membranes. Our research identifies Caenorhabditis elegans homologue of synaptosomal-associated protein 29 (SNAP-29) as an essential regulator of membrane trafficking in polarized intestinal cells of living animals. We show that a depletion of SNAP-29 blocks yolk secretion and targeting of apical and basolateral plasma membrane proteins in the intestinal cells and results in a strong accumulation of small cargo-containing vesicles. The loss of SNAP-29 also blocks the transport of yolk receptor RME-2 to the plasma membrane in nonpolarized oocytes, indicating that its function is required in various cell types. SNAP-29 is essential for embryogenesis, animal growth, and viability. Functional fluorescent protein–tagged SNAP-29 mainly localizes to the plasma membrane and the late Golgi, although it also partially colocalizes with endosomal proteins. The loss of SNAP-29 leads to the vesiculation/fragmentation of the Golgi and endosomes, suggesting that SNAP-29 is involved in multiple transport pathways between the exocytic and endocytic organelles. These observations also suggest that organelles comprising the endomembrane system are highly dynamic structures based on the balance between membrane budding and fusion and that SNAP-29–mediated fusion is required to maintain proper organellar morphology and functions.
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Dubé, Mathieu, Loïc Etienne, Maximilian Fels, and Margaret Kielian. "Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes." Journal of Virology 90, no. 14 (April 27, 2016): 6303–13. http://dx.doi.org/10.1128/jvi.00634-16.
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ABSTRACTThe E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH.IMPORTANCERubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment.
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Zaitseva, Elena, Aditya Mittal, Diane E. Griffin, and Leonid V. Chernomordik. "Class II fusion protein of alphaviruses drives membrane fusion through the same pathway as class I proteins." Journal of Cell Biology 169, no. 1 (April 4, 2005): 167–77. http://dx.doi.org/10.1083/jcb.200412059.
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Viral fusion proteins of classes I and II differ radically in their initial structures but refold toward similar conformations upon activation. Do fusion pathways mediated by alphavirus E1 and influenza virus hemagglutinin (HA) that exemplify classes II and I differ to reflect the difference in their initial conformations, or concur to reflect the similarity in the final conformations? Here, we dissected the pathway of low pH–triggered E1-mediated cell–cell fusion by reducing the numbers of activated E1 proteins and by blocking different fusion stages with specific inhibitors. The discovered progression from transient hemifusion to small, and then expanding, fusion pores upon an increase in the number of activated fusion proteins parallels that established for HA-mediated fusion. We conclude that proteins as different as E1 and HA drive fusion through strikingly similar membrane intermediates, with the most energy-intensive stages following rather than preceding hemifusion. We propose that fusion reactions catalyzed by all proteins of both classes follow a similar pathway.
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Dawe, Sandra, Jennifer A. Corcoran, Eileen K. Clancy, Jayme Salsman, and Roy Duncan. "Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein." Journal of Virology 79, no. 10 (May 15, 2005): 6216–26. http://dx.doi.org/10.1128/jvi.79.10.6216-6226.2005.
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ABSTRACT Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic Nexoplasmic/Ccytoplasmic topology. This topology results in the translocation of the smallest ectodomain (∼20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.
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Mahal, Lara K., Sonia M. Sequeira, Jodi M. Gureasko, and Thomas H. Söllner. "Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I." Journal of Cell Biology 158, no. 2 (July 15, 2002): 273–82. http://dx.doi.org/10.1083/jcb.200203135.
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Ñeurotransmitter release requires the direct coupling of the calcium sensor with the machinery for membrane fusion. SNARE proteins comprise the minimal fusion machinery, and synaptotagmin I, a synaptic vesicle protein, is the primary candidate for the main neuronal calcium sensor. To test the effect of synaptotagmin I on membrane fusion, we incorporated it into a SNARE-mediated liposome fusion assay. Synaptotagmin I dramatically stimulated membrane fusion by facilitating SNAREpin zippering. This stimulatory effect was topologically restricted to v-SNARE vesicles (containing VAMP 2) and only occurred in trans to t-SNARE vesicles (containing syntaxin 1A and SNAP-25). Interestingly, calcium did not affect the overall fusion reaction. These results indicate that synaptotagmin I can directly accelerate SNARE-mediated membrane fusion and raise the possibility that additional components might be required to ensure tight calcium coupling.
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Jeetendra, E., Clinton S. Robison, Lorraine M. Albritton, and Michael A. Whitt. "The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion." Journal of Virology 76, no. 23 (December 1, 2002): 12300–12311. http://dx.doi.org/10.1128/jvi.76.23.12300-12311.2002.
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ABSTRACT Recently we showed that the membrane-proximal stem region of the vesicular stomatitis virus (VSV) G protein ectodomain (G stem [GS]), together with the transmembrane and cytoplasmic domains, was sufficient to mediate efficient VSV budding (C. S. Robison and M. A. Whitt, J. Virol. 74:2239-2246, 2000). Here, we show that GS can also potentiate the membrane fusion activity of heterologous viral fusion proteins when GS is coexpressed with those proteins. For some fusion proteins, there was as much as a 40-fold increase in syncytium formation when GS was coexpressed compared to that seen when the fusion protein was expressed alone. Fusion potentiation by GS was not protein specific, since it occurred with both pH-dependent as well as pH-independent fusion proteins. Using a recombinant vesicular stomatitis virus encoding GS that contained an N-terminal hemagglutinin (HA) tag (GSHA virus), we found that the GSHA virus bound to cells as well as the wild-type virus did at pH 7.0; however, the GSHA virus was noninfectious. Analysis of cells expressing GSHA in a three-color membrane fusion assay revealed that GSHA could induce lipid mixing but not cytoplasmic mixing, indicating that GS can induce hemifusion. Treatment of GSHA virus-bound cells with the membrane-destabilizing drug chlorpromazine rescued the hemifusion block and allowed entry and subsequent replication of GSHA virus, demonstrating that GS-mediated hemifusion was a functional intermediate in the membrane fusion pathway. Using a series of truncation mutants, we also determined that only 14 residues of GS, together with the VSV G transmembrane and cytoplasmic tail, were sufficient for fusion potentiation. To our knowledge, this is the first report which shows that a small domain of one viral glycoprotein can promote the fusion activity of other, unrelated viral glycoproteins.
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Huang, Xiaofang, Xin Zhou, Xiaoyu Hu, Amit S. Joshi, Xiangyang Guo, Yushan Zhu, Quan Chen, William A. Prinz, and Junjie Hu. "Sequences flanking the transmembrane segments facilitate mitochondrial localization and membrane fusion by mitofusin." Proceedings of the National Academy of Sciences 114, no. 46 (November 1, 2017): E9863—E9872. http://dx.doi.org/10.1073/pnas.1708782114.
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Mitochondria constantly divide and fuse. Homotypic fusion of the outer mitochondrial membranes requires the mitofusin (MFN) proteins, a family of dynamin-like GTPases. MFNs are anchored in the membrane by transmembrane (TM) segments, exposing both the N-terminal GTPase domain and the C-terminal tail (CT) to the cytosol. This arrangement is very similar to that of the atlastin (ATL) GTPases, which mediate fusion of endoplasmic reticulum (ER) membranes. We engineered various MFN-ATL chimeras to gain mechanistic insight into MFN-mediated fusion. When MFN1 is localized to the ER by TM swapping with ATL1, it functions in the maintenance of ER morphology and fusion. In addition, an amphipathic helix in the CT of MFN1 is exchangeable with that of ATL1 and critical for mitochondrial localization of MFN1. Furthermore, hydrophobic residues N-terminal to the TM segments of MFN1 play a role in membrane targeting but not fusion. Our findings provide important insight into MFN-mediated membrane fusion.
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Mendonsa, Rima, and JoAnne Engebrecht. "Phosphatidylinositol-4,5-Bisphosphate and Phospholipase D-Generated Phosphatidic Acid Specify SNARE-Mediated Vesicle Fusion for Prospore Membrane Formation." Eukaryotic Cell 8, no. 8 (June 5, 2009): 1094–105. http://dx.doi.org/10.1128/ec.00076-09.
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ABSTRACT The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of proteins is required for eukaryotic intracellular membrane fusions. Vesicle fusion for formation of the prospore membrane (PSM), a membrane compartment that forms de novo during yeast sporulation, requires SNARE function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], and the activity of the phospholipase D (PLD) Spo14p, which generates phosphatidic acid (PA). The SNARE syntaxin Sso1p is essential for PSM production while the functionally redundant homolog in vegetative growth, Sso2p, is not. We demonstrate that Sso1p and Sso2p bind similarly in vitro to PA or phosphoinositide-containing liposomes and that the conserved SNARE (H3) domain largely mediates PA-binding. Both green fluorescent protein-Sso fusion proteins localize to the developing PSM in wild-type cells and to the spindle pole body in spo14Δ cells induced to sporulate. However, the autoregulatory region of Sso1p binds PI(4,5)P2-containing liposomes in vitro with a greater ability than the equivalent region of Sso2p. Overexpression of the phosphatidylinositol-4-phosphate 5-kinase MSS4 in sso1Δ cells induced to sporulate stimulates PSM production; PLD activity is not increased under these conditions, indicating that PI(4,5)P2 has roles in addition to stimulating PLD in PSM formation. These data suggest that PLD-generated PA and PI(4,5)P2 collaborate at multiple levels to promote SNARE-mediated fusion for PSM formation.
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Yamamoto, Mizuki, Shutoku Matsuyama, Xiao Li, Makoto Takeda, Yasushi Kawaguchi, Jun-ichiro Inoue, and Zene Matsuda. "Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay." Antimicrobial Agents and Chemotherapy 60, no. 11 (August 22, 2016): 6532–39. http://dx.doi.org/10.1128/aac.01043-16.
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ABSTRACTMiddle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressingRenillaluciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infectionin vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.
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Weber, Thomas, Francesco Parlati, James A. McNew, Robert J. Johnston, Benedikt Westermann, Thomas H. Söllner, and James E. Rothman. "Snarepins Are Functionally Resistant to Disruption by Nsf and αSNAP." Journal of Cell Biology 149, no. 5 (May 29, 2000): 1063–72. http://dx.doi.org/10.1083/jcb.149.5.1063.
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SNARE (SNAP [soluble NSF {N-ethylmaleimide–sensitive fusion protein} attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.
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Klupp, Barbara G., Ralf Nixdorf, and Thomas C. Mettenleiter. "Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion." Journal of Virology 74, no. 15 (August 1, 2000): 6760–68. http://dx.doi.org/10.1128/jvi.74.15.6760-6768.2000.
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ABSTRACT A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.
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Shmulevitz, Maya, Jayme Salsman, and Roy Duncan. "Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation." Journal of Virology 77, no. 18 (September 15, 2003): 9769–79. http://dx.doi.org/10.1128/jvi.77.18.9769-9779.2003.
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ABSTRACT Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo- or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.
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Adrien, Vladimir, Hugo Fumat, Cédric Tessier, Philippe Nuss, and David Tareste. "T202. THE EFFECT OF ANTIPSYCHOTIC DRUGS ON MEMBRANE FUSION: AN IN VITRO STUDY." Schizophrenia Bulletin 46, Supplement_1 (April 2020): S308—S309. http://dx.doi.org/10.1093/schbul/sbaa029.762.
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Abstract Background Common clinical use of antipsychotics (AP) drugs shows that their therapeutic mode of action still needs further clarification although it is admitted that the Dopamine receptor D2 (D2R) antagonism plays a significant role. For instance, clozapine (CLOZ) - which is known to be the most effective AP in treating schizophrenic symptoms - has strikingly the lowest D2R antagonism. Non direct receptor-related effects might thus be involved in the activity of AP at the synapse level. AP, as well as neurotransmitters, are mostly lipophilic and insert within membranes. This characteristic is of interest as a significant proportion of schizophrenic patients has specific and abnormal membrane lipid composition. This possible proxy of the disease biotype can participate in the disease’s physiopathology but also be critical for the effect of AP drugs. We hypothesize that AP insertion into lipid membranes also contribute to their therapeutic effect. AP-induced modifications of synaptic membranes biophysics are likely to influence neurotransmission. In this study, we focus on the effect of AP on membrane fusion, a crucial step for the exocytosis of neurotransmitters. Methods Liposomes modelling synaptic vesicles were reconstituted in saline buffer. Two standard ternary and quaternary lipid mixtures have been studied: phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine (PC:PE:PS) [65:25:10] and the synaptic-like PC:PE:PS:sphingomyelin:cholesterol (PC:PE:PS:SM:CHOL) [25:25:10:10:30]. Some liposomes were protein-free and others were functionalized with Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) proteins, which trigger in vivo the fusion of synaptic vesicles with the pre-synaptic plasma membrane. The liposome size was checked by Dynamic Light Scattering. Insertion of AP within the membrane was checked by second derivative spectroscopy. Fusion was measured by Fluorescence Resonance Energy Transfer in the absence or presence of CLOZ or chlorpromazine (CPZ) at various lipid:AP ratios (10:1 to 100000:1). Protein-free liposomes were fused with Polyethylene glycol (PEG) and SNARE liposomes through the action of cognate SNARE proteins residing in their membrane. Results Liposomes of the same lipid composition were of the same size, with no effect of the addition of AP drugs at various concentrations. Molar partition coefficient of AP drugs within the membrane of protein-free liposomes was approximately 70–85%. CPZ or CLOZ inhibited the fusion of PC:PE:PS liposomes by about 20–40%. When liposomes were synaptic-like (PC:PE:PS:SM:CHOL), the inhibition of fusion by AP drugs reached 50%. CLOZ also inhibited SNARE-mediated fusion of PC:PE:PS liposomes by about 30%. This effect on SNARE-mediated fusion was not observed with CPZ. Discussion Altogether, these results, despite preliminary, could help to understand partially a non direct receptor-related effect of antipsychotics. Indeed, these drugs also seem to modify membrane dynamics at the synapse level. This seems to be particularly the case of CLOZ on SNARE-mediated fusion and could explain its specific therapeutic efficiency.
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Chan, Ka Man Carmen, Ashley L. Arthur, Johannes Morstein, Meiyan Jin, Abrar Bhat, Dörte Schlesinger, Sungmin Son, Donté A. Stevens, David G. Drubin, and Daniel A. Fletcher. "Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion." Proceedings of the National Academy of Sciences 118, no. 1 (December 21, 2020): e2007526118. http://dx.doi.org/10.1073/pnas.2007526118.
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Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP–mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.
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Yu, Haijia, Yinghui Liu, Daniel R. Gulbranson, Alex Paine, Shailendra S. Rathore, and Jingshi Shen. "Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites." Proceedings of the National Academy of Sciences 113, no. 16 (April 4, 2016): 4362–67. http://dx.doi.org/10.1073/pnas.1517259113.
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Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca2+, allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca2+. In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum–plasma membrane contact sites, are Ca2+-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca2+. E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca2+-bound C2 domains. Strikingly, the Ca2+-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca2+ sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca2+ regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca2+-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca2+ regulation.