In Vivo Analysis of Membrane Fusion

Mark T Palfreyman, University of Utah, Salt Lake City, UT, USA
Erik M Jorgensen, University of Utah, Salt Lake City, UT, USA

Published online: March 2009

DOI: 10.1002/9780470015902.a0020891

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.

Key concepts

  • Fusion proteins are thought to lead membranes through the common set of lipid intermediates: lipid stalk, hemifusion diaphragm and pore formation.
  • There are three known classes of viral fusogens: type I, II and III.
  • Type I and II are structurally unique yet undergo similar structural rearrangements during membrane fusion.
  • Viral fusion proteins are initially present only on a single membrane and must insert a fusion peptide into the target membrane to accomplish fusion.
  • SNAREs are used throughout the secretory pathway.
  • SNAREs are present on both membranes destined to fuse.
  • SNAREs and viral fusion proteins play an active role in all steps of the fusion process.
  • Multimerization of viral fusion proteins and SNARE proteins is needed for fusion to occur.
  • The fusogens used in cell–cell fusion are not evolutionarily conserved.
  • The coordinated action of Fzo1 and Mgm1 is needed to fuse the inner and outer membranes of mitochondria.

Keywords: membrane fusion; virus; SNARE; lipid; fusogen

Figure 1. Common lipid intermediates during fusion. The proximal leaflets of the plasma bilayers are brought into close proximity forming the hourglass-like structure known as a lipid stalk. This stalk is then expanded forming a hemifusion diaphragm. In the hemifusion diaphragm the distal leaflets of the bilayer are now in direct contact. A rupture in the hemifusion diaphragm leads to the initial opening of a fusion pore that is then expanded leading to full fusion. The insert shows the shape of lysophosphatidylcholine (LPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). LPC induces positive curvature whereas PE induces negative curvature. Adding lipids with positive curvature to the proximal leaflets inhibits stalk formation (red lipid – ‘stalk’ and ‘two membranes’ steps). Adding lipids with positive curvature to the distal leaflets promotes fusion (red lipid – ‘pore step’). Arrowheads point to areas where the lipid must adopt a net negative curvature.
Figure 2. Viral fusion using the influenza HA fusion protein as the primary example. Structural rearrangements of the E fusion protein from flavivirus are shown to illustrate commonalities and differences between type I and type II fusion proteins. (a) The influenza virus enters the host by being taken up through the endocytic pathway. Upon endocytosis the acidic environment in the endosome activates the HA fusion proteins and the viral capsid is released into the cytoplasm. Structural rearrangements are described in detail in (c), then lead to the fusion of viral and host membranes releasing the contents of the influenza virus into the cytoplasm. (b) The structures of the three classes of viral fusion proteins vary widely, yet all undergo common rearrangements that catalyse fusion (compare (c) and (d)). The post-fusion structures of all three classes of viral fusion proteins are shown. The transmembrane domains and fusion peptides are not shown. The examples of each class are: HIV gp41 (type I), Flavivirus fusion protein E (type II) and VSV glycoprotein G (type III). HA is a type I fusion protein. (c) All viral fusion reactions are catalysed by common rearrangements in the viral fusion proteins. Here HA and E are taken as the examples for comparison. Initially, HA and E are present only on the viral membrane (1). Upon activation, in this case by low pH, the fusion peptide (in red) is exposed and inserted into the host membrane (2). In the case of the type II E fusion protein, E monomers come together to form a trimer after low pH activation. By contrast, the type I HA fusion proteins are thought to exist as trimers before activation. After activation, the viral and host membranes are then brought into close proximity (3). The zippering of the HA and E proteins induces lipid stalk formation in which the proximal leaflets of the membranes are fused (4). Full zippering induces pore formation, in which the distal leaflets have now become one (5). The post-fusion HA and E proteins are left on the membranes of the host having accomplished their function – catalysing the release of the viral genome into the host cell (6). Structures illustrated in (b) are adapted from Weissenhorn et al. (2007) Virus membrane fusion. FEBS Letters 581: 2150–2155.
Figure 3. SNARE-based fusion using neurotransmission as the example. (a) Synaptic vesicles fuse at synapses and release neurotransmitters into the synaptic cleft. SNARE proteins are present on synaptic vesicles (synaptobrevin – blue) and on the plasma membrane (syntaxin – red and SNAP25 – green). Structural rearrangements in the SNARE proteins are described in detail in (d). (b) The structure of the fully assembled SNARE complex. The example shown is of the SNAREs used in neurotransmission; however, fusion reactions in the secretory pathway all use related SNARE proteins. These proteins form a four-helix bundle during fusion – the so-called core complex. Syntaxin contains an additional inhibitory domain known as the Habc domain. (c) The four SNARE family members are characterized by conserved residues that face into the centre of the four-helix bundle. Illustrated is a crosswise slice through the core complex showing the interactions between arginine on the R-SNARE (synaptobrevin) and the three glutamines of the Qa-SNARE (syntaxin) and the Qbc-SNARE (SNAP25). In many fusion complexes the Qb and Qc helices are located on separate proteins rather than on a single molecule as is the case with SNAP25. (d) Unlike viruses, SNAREs are initially located on both membranes destined to fuse (1). Before assembly into the core complex, the SNARE domains are unstructured except for syntaxin which adopts a closed confirmation where the inhibitory Habc domain folds over the SNARE motif. The plasma membrane SNAREs are thought to form an acceptor complex in which syntaxin and SNAP25 partially assemble (2). Synaptobrevin then joins the complex, by making initial contact at the N-terminal membrane-distal portion (3). The SNAREs become progressively more structured in an N- to C-terminal direction as they zipper up. The zippering of the SNAREs pulls the proximal leaflets of the membranes together so that they fuse and form a lipid stalk (4). The continued zippering opens the initial fusion pore (5) – the distal leaflets of the membranes have now become one. Post-fusion, all of the SNARE proteins are located in the plasma membrane (6).
Figure 4. Cell–cell fusion reactions are catalysed by an assortment of unique fusion proteins. EFF-1 is used in C. elegans during the formation of the hypodermis. EFF-1 mediates homotypic fusion – it must be present on both membranes destined to fuse. It has been demonstrated to be necessary and sufficient for fusion. The best candidates for sperm–egg fusion are Izumo localized on sperm and CD9 localized on egg. Izumo encodes a member of the immunoglobulin superfamily. CD9 is a member of the tetraspanin superfamily. It is not known how Izumo and CD9 might catalyse fusion. Yeast haploid cells fuse during mating. Molecules suggested to mediate yeast mating cell fusion are: Prm1p, Fig1p and Fus1p. Prm1p is multipass membrane protein whose expression is induced by the mating pheromone. Fus1p is a single pass membrane protein in whose absence the fusion pore opening is delayed and inefficient. Fig1p is a multipass membrane protein. Fig1p fusion defects can be rescued by increasing calcium concentration. Placenta development requires cell–cell fusion in the syncytiotrophoblast layer. This layer forms the interface between fetus and mother. Fusion of the syncytiotrophoblast is reduced by antibodies against syncytins. Syncytins are fusogens from an endogenous retrovirus expressed specifically in the syncytiotrophoblast layer.
Figure 5. Outside of the secretory pathway, organelle fusion is not mediated by the SNARE proteins. These non-SNARE fusion reactions include those that maintain mitochondria and peroxisomes. Mitochondria are unique in that they contain an inner and outer membrane whose fusion must be coordinated. Fusion of the outer membrane is catalysed by fuzzy onions (Fzo1p), known as mitofusins in mammals. Fzo1p contains two transmembrane domains anchoring it in the outer membrane. The transmembrane domains are separated by a loop that extends into the space between outer and inner membranes and may play a role in coordinating the fusion of these two membranes. Facing into the cytoplasm, Fzo1p contains a GTPase domain (green oval) and -helices (red rods) that form coiled-coils between two mitochondria destined to fuse. Inner membrane fusion is catalysed by Mgm1p, known as Opa1 in mammals. The dynamin-related Mgm1p contains a GTPase domain (purple oval) and is peripherally localized to the inner membrane. In yeast the coordination of outer and inner membrane fusion might be accomplished by Ugo1p. Peroxisomes also do not appear to use SNAREs for membrane fusion. Two triple A+ ATPases, related to NSF, have phenotypes consistent with a membrane fusion defect. The triple A+ ATPases, Pex1p and Pex6p are localized to peroxisomes via Pex26p. Currently there are no specific models for their role in fusion.
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 Further Reading
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General Virology | Cell Membranes | Techniques in Cell Biology | Membrane Proteins | Lipids and Membrane Structure | Virus Structure and Function
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Article Title: In Vivo Analysis of Membrane Fusion
 
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Palfreyman, Mark T, and Jorgensen, Erik M(Mar 2009) In Vivo Analysis of Membrane Fusion. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0020891]