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Membrane Fusion: Anchors aweigh
MEMBRANE FUSION
TOON STEGMANN
Anchors aweigh A recent study using a version of the influenza virus hemagglutinin protein in which its membrane anchor was replaced by a lipid tail provides new insights into how proteins can mediate the fusion of membranes. Sometimes, in science, you have to go a long way around a problem to solve it. Take, for example, membrane fusion, which is involved in processes such as intracellular protein transport, exocytosis, fertilization and the entry of enveloped animal viruses into their host cells. Fusion is accomplished by the merging of two lipid bilayers, but, as this merging is strictly regulated and timed, it must be mediated by proteins. It is difficult to study how proteins persuade lipids to mix, because the elegant techniques that are available to study proteins, such as genetic engineering, cannot be used for lipids. Moreover, lipid bilayers are very dynamic structures; lipid molecules are too small to be seen in the electron microscope, and only a minor fraction of the lipids in a biological membrane is involved in fusion at any given time, ruling out most spectroscopic techniques. Lipid behaviour during membrane fusion has, therefore, been studied either by biophysical techniques in pure lipid model systems, or by indirect means in the case of protein-mediated membrane fusion, such as mutating the sequences of fusion proteins, observing the effect on fusion, and trying to deduce the effect on lipids. Now, Kemble et al. [1] have directly observed a change in lipid behavior in response to the modification of a fusion-mediating protein, providing evidence for one mechanism of membrane fusion. The recent work uses the most extensively studied fusion protein, the hemagglutinin of influenza virus.
This virus first binds to its receptor, sialic acid, on the surface of the host cell, and is then internalized by endocytosis and taken into the endosomal pathway. The low pH inside endosomes induces a conformational change in hemagglutinin, leading to fusion between the viral envelope and the endosomal membrane. Fusion of these two membranes results in the release of the viral RNA into the cytoplasm of the host cell and hence infection. Hemagglutinin is an integral membrane protein which is synthesized as a precursor, HAO, that assembles into a trimer shortly after synthesis. HAO is then proteolytically cleaved to produce two subunits, HA1 and HA2, which remain linked by a single disulfide bridge. HA2 provides an extensively palmitoylated carboxy-terminal membrane anchor and a short cytoplasmic tail. The amino terminus of HA2 is hydrophobic and is required for fusion; it is therefore often referred to as the 'fusion peptide'. The structure of the neutral-pH form of the ectodomain of hemagglutinin, as determined by X-ray crystallography, shows that the fusion peptide is hidden in the stem of the trimer (Fig. la), which consists mainly of HA2 [2]. HA1 makes up most of the membrane-distal domain of the protein and provides the receptor-binding site. There is no fusion-active that low pH involves two
consensus as to the conformation of the form of hemagglutinin at low pH. It seems induces a conformational change which steps: first, the fusion peptide is exposed
Fig. 1. Conformational changes in hemagglutinin. (a) Hemagglutinin trimer at neutral pH (structure of hemagglutinin from Wilson et al. [21, cartoon after Bentz [10]). (b) Fusion-active low pH conformation postulated in [8]. (c) Postulated final fusion-inactive low-pH conformation. A single monomer in (a) is outlined by a thick black line.
© Current Biology 1994, Vol 4 No 6
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Current Biology 1994, Vol 4 No 6 (Fig. lb), and then the globular heads of the trimer dissociate (Fig. c) (reviewed by White [3]). A very different model for the conformational change has been proposed recently [4] but remains to be confirmed experimentally. The extent to which the globular heads dissociate in the fusion-active conformation is disputed. If two additional cysteines are engineered into HA1, causing monomers of HA1 to cross-link to adjacent monomers via disulfide bridges at the tips of the trimer, exposure of the fusion peptide is impaired and there is no fusion [5,6]. On the other hand, the hemagglutinins from several strains of virus mediate fusion without separation of the globular heads [7]. Those hemagglutinins that do undergo extensive dissociation of the head domains at 37 C can still mediate fusion at 0 C, even though it can be shown, using monoclonal antibodies that recognize epitopes in the tip of the trimer, that head dissociation does not occur at this temperature [8]. When a preparation of virus with these characteristics is taken to low pH at 37°C, in the absence of membranes with which to fuse, the globular heads of hemagglutinin separate. Once the globular heads separate, the molecule quickly becomes inactivated. If virus treated in this way is subsequently added to target membranes, it is no longer able to induce fusion. These results suggest that limited changes at the tip of the trimer are required for fusion, and that a transient intermediate (Fig. lb) represents the fusionactive form of the molecule, whereas the completely dissociated form of hemagglutinin (Fig. c) has no fusion capacity. How could the conformational change in hemagglutinin lead to membrane fusion? Different experimental approaches have led to a plethora of models for hemagglutinin-lipid interactions during fusion. Most workers agree that, after the conformational change, the fusion peptides immediately insert into the target membrane for fusion (although there is not universal agreement on this point; see below). Experiments using photoactivatable lipid analogues in the target membrane have demonstrated that insertion of the fusion peptide precedes the actual merging of the membranes [9]. Furthermore, it is widely believed that fusion is caused by fusion complexes, which are higher order multimers of hemagglutinin trimers, and that fusion is induced by transient intermediate protein-lipid structures. Three of the main models for such intermediates are shown in Figure 2 (in parts a, b and d). The first model (Fig. 2a) [10] differs from most others in not assuming that the fusion peptides insert into the target membrane. Instead, it proposes that the fusion complex consists of a ring of hemagglutinin molecules, with fusion peptides lining the inside of the complex and providing a hydrophobic surface for contact with the lipid tails of an inverted micelle, a sphere or ovoid of lipids that have their hydrophobic tails pointing outward and their hydrophilic heads facing an aqueous interior. Inverted micelles were found to be fusion intermediates in several model systems used for studying
membrane fusion. The micelles would initiate mixing of the lipids from the outer membrane leaflets, then cause rupture of the inner membrane leaflets at the site of micelle formation, resulting in fusion. But it has been shown that hemagglutinin-mediated membrane fusion can take place under conditions in which inverted micelles cannot form [11], and there is no enhancement of fusion under conditions that promote their formation [12]. These findings suggest that the model shown in Figure 2a is probably incorrect. The second model (Fig. 2b) does not make specific assumptions about the conformation of hemagglutinin or the structure of the lipids, but proposes that a proteinaceous 'fusion pore' is formed initially. The model is based on electrophysiological measurements of the fusion of voltage-clamped hemagglutinin-expressing cells with erythrocytes labelled with a fluorescent lipid probe [13]. Upon fusion, a sudden increase in capacitance of the hemagglutinin-expressing cell was recorded, due to the increase in membrane surface area - but the fluorescent lipids diffused into the membrane of the hemagglutinin-expressing cell only much later. This means that an aqueous connection, the 'fusion pore', is formed between the two fusing cells and that it does not initially allow lipid flow. It was therefore proposed that an entirely proteinaceous fusion complex, consisting of a ring of hemagglutinin molecules, would form a water-tight pore through both membranes (Fig. 2b). Following an initial jump, the capacitance was found to 'flicker' a few times; it was therefore suggested [13] that the pore initially opened and closed repeatedly. Subsequently, the pore would widen gradually, somehow acquiring lipids sideways, and initiating fusion. In the third model [3,14,15] (Fig. 2d), the fusion-active form of hemagglutinin would be similar to that shown in Figure lb. At neutral pH, the fusion peptide is 30 A away from the viral membrane but 100 A from the tip of the molecule (Fig. la). Thus, to explain the observed [9,16] insertion of fusion peptides into the target membrane, it is assumed that hemagglutinin must bend (Fig. 2d) [8]. In this model, the fusion peptides in the bent form of the molecule could insert into the viral membrane as well as into the target membrane. Fusioncomplex formation would bring fusion peptides together, and the resulting high local concentration of fusion peptides could create a fault in the target membrane [15]. Beginning at the fault, rotation of the peptides could give rise to the formation of lipidic intermediate structures known as stalks, which are spool-shaped and composed of fused outer membrane leaflets of the viral and target membrane bilayers (Fig. 2d, center) [15]. Rupture of the inner membrane leaflets will then lead to complete fusion giving rise to a lipid-lined fusion pore (Fig. 2d, bottom). The pore may be restrained initially, for example by the inserted fusion peptides, and could revert to a stalk intermediate and back again to a pore repeatedly, which could explain the capacitance flicker mentioned
DISPATCH
Fig. 2. Proposed mechanisms of membrane fusion. (a) Fusion via inverted micelles (after Bentz [10]). (b) One possible interpretation of a proteinaceous fusion pore. (c) Hemifusion (after Kemble et al. [1]). (d) Stalk model for membrane fusion (after Siegel [15] and Stegmann and Helenius [14]): insertion of the fusion peptide into the target and viral membrane; stalk formation; and fusion.
previously [13]. The transmembrane anchor of hemagglutinin could act to lower the activation energy of stalk formation [15] and, if concentrated in the fusion complex, might create a fault in the viral membrane. It has also been suggested that the ectodomain of hemagglutinin cannot bend relative to its membrane anchor: if this is the case, then hemagglutinin would have to tilt as a whole, deforming the viral membrane and creating a bulge that might lower the barriers to membrane fusion [171. To investigate the role of the membrane anchor of hemagglutinin in fusion, Kemble et al. [1] replaced the transmembrane domain of hemagglutinin with a glycosylphosphatidylinositol (GPI) tail, a lipid anchor that inserts into the outer membrane leaflet but not into the inner leaflet. The GPI-tailed hemagglutinin was expressed on the surface of a cell, and its ability to induce. fusion with erythrocytes that contained either water-soluble fluorescent probes or lipid-embedded fluorescent probes was investigated. Surprisingly, the water-soluble probes were not delivered to the cytoplasm of the GPI-hemagglutinin-expressing cell, but transfer of the lipid probes was similar in kinetics and efficiency to that seen with wild-type, non-GPIanchored hemagglutinin. This must mean that the outer
membrane leaflets of the erythrocyte and the GPI-hemagglutinin-expressing cell mixed, but the inner membrane leaflets did not (Fig. 2c), a situation found sporadically in model systems of membrane fusion and termed 'hemifusion'. It therefore appears that hemifusion mediated by GPI-hemagglutinin produces the equivalent of a stalk-like intermediate (see Fig. 2d, center). If GPI-hemagglutinin suffices to induce stalk formation but not full fusion, then the membrane and anchor domains of hemagglutinin must be responsible for rupture and fusion of the inner membrane leaflet of the plasma membrane in which hemagglutinin is embedded [1]. The lipid-lined fusion pore of the stalk model would appear to be incompatible with the results of the electrophysiological measurements, which indicated that the initial pore is proteinaceous because it is formed before the lipids have mixed (Fig. 2b) [13]. But it could be that the ring of hemagglutinin membrane-anchors in the fusion complex acts to prevent, or significantly slow, the flow of lipids through the stalk, in the manner of a tight junction. The GPI tails would not pose such a barrier [1]. As a result of the high curvature of the lipids in a stalk, it is hard to imagine that hemifusion involves
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a stably arrested stalk structure. It is more likely that a dynamic equilibrium between the stage preceding stalk formation (Fig. 2d, top) and stalk structures (Figs 2c and 2d, center) exists. If this is the case, with luck, one may be able to detect fluorescent lipid probes being transferred from an erythrocyte to a GPI-hemagglutinin cell in bursts. And, if stalks are formed all the time, the chances of being able directly to see stalks between the two cells by electron microscopy should be much better. It is also possible that stalks are formed just once, leading to the transfer of lipids, and then the hemagglutinin is inactivated. While these exciting questions can now be addressed experimentally, the results of the GPI-hemagglutinin experiments strongly suggest that fusion involves a stalk-like intermediate, explaining how, at least in this case, a protein gets lipid bilayers to mix. References 1. KEMBLE GW, DANIELI T, WHITE JM: Lipid-anchored influenza 2. 3. 4. 5.
6.
hemagglutinin promotes hemifusion, not complete fusion. Cell 1994, 76:383-391. WILSON IA, SKEHEL JJ, WILEY DC: Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 1981, 289:366-375. WHITE JM: Membrane fusion. Science 1992, 258:917-924. CARR CM, KIM PS: A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell 1993, 73:823-832. GODLEY L, PFEIFER J, STEINHAUER D, ELY B, SHAW G, KAUFMAN R, SUCHANEK E, PABO C, SKEHEL JJ, WILEY DC, WHARTON S: Introduction of intersubunit disulfide bonds in the membranedistal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell 1992, 68:635-645. KEMBLE GW, BODIAN DL, ROSE J, WILSON IA, WHITE JM: Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin. J Virol 1992, 66:4940-4950.
7.
PURI A, BOOY FP, DOMS RW, WHITE JM, BLUMENTHAL R:
Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment. J Virol 1990, 64:3824-3832. 8.
STEGMANN T, WHITE JM, HELENIUS A: Intermediates in influenza
induced membrane fusion. EMBOJ 1990, 9:4231-4241. 9.
STEGMANN T, DELFINO JM, RICHARDS FM, HELENIUS A: The
HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion. J Biol Chem 1991, 266:18404-18410. 10. BENTZ J, ELLENS H, ALFORD D: Architecture of the influenza
hemagglutinin fusion site. In Vtralfusion mechanisms. Edited by Bentz J. Boca Raton: CRC Press; 1993:163-199. 11. STEGMANN T: Influenza hemagglutinin-mediated membrane fusion does not involve inverted phase lipid intermediates. J Bol Chem 1993, 268:1716-1722. 12. ALFORD D, ELLENS H, BENTZ J: Fusion of influenza virus with
sialic acid bearing target membranes. Biochemistry 1994, 33:1977-1987. 13. TSE FW, IWATA A, ALMERS W: Membrane flux through the pore
formed by a fusogenic viral envelope protein during cell fusion. J Cell Biol 1993, 121:543-552. 14. STEGMANN T, HELENIUS A: Influenza virus fusion: from models toward a mechanism. In Viral Fusion Mechanisms. Edited by Bentz J. Boca Raton: CRC press; 1993:89-111. 15. SIEGEL DP: Modeling protein-induced fusion mechanisms: insights from the relative stability of lipidic structures. In Viral Fusion Mechanisms. Edited by Bentz J. Boca Raton: CRC Press; 1993:475-512. 16. TSURUDOME M, GLOCK R, GRAF R, FALCHETTO R, SCHALLER U,
BRUNNER J: Mechanism of influenza induced membrane fusion: membrane insertion of the 'fusion' peptide of hemagglutinin. JBiol Chem 1992, 267:20225-20232. 17. WILSCHUT J, BRON R: The influenza hemagglutinin: membrane fusion activity in intact virions and reconstituted virosomes. In Viralfusion mechanisms. Edited by Bentz J. Boca Raton: CRC Press; 1993:133-161.
Toon Stegmann, Department of Biophysical Chemistry, Biozentrum of the University of Basel, CH 4056, Basel, Switzerland.
TOON STEGMANN
Anchors aweigh A recent study using a version of the influenza virus hemagglutinin protein in which its membrane anchor was replaced by a lipid tail provides new insights into how proteins can mediate the fusion of membranes. Sometimes, in science, you have to go a long way around a problem to solve it. Take, for example, membrane fusion, which is involved in processes such as intracellular protein transport, exocytosis, fertilization and the entry of enveloped animal viruses into their host cells. Fusion is accomplished by the merging of two lipid bilayers, but, as this merging is strictly regulated and timed, it must be mediated by proteins. It is difficult to study how proteins persuade lipids to mix, because the elegant techniques that are available to study proteins, such as genetic engineering, cannot be used for lipids. Moreover, lipid bilayers are very dynamic structures; lipid molecules are too small to be seen in the electron microscope, and only a minor fraction of the lipids in a biological membrane is involved in fusion at any given time, ruling out most spectroscopic techniques. Lipid behaviour during membrane fusion has, therefore, been studied either by biophysical techniques in pure lipid model systems, or by indirect means in the case of protein-mediated membrane fusion, such as mutating the sequences of fusion proteins, observing the effect on fusion, and trying to deduce the effect on lipids. Now, Kemble et al. [1] have directly observed a change in lipid behavior in response to the modification of a fusion-mediating protein, providing evidence for one mechanism of membrane fusion. The recent work uses the most extensively studied fusion protein, the hemagglutinin of influenza virus.
This virus first binds to its receptor, sialic acid, on the surface of the host cell, and is then internalized by endocytosis and taken into the endosomal pathway. The low pH inside endosomes induces a conformational change in hemagglutinin, leading to fusion between the viral envelope and the endosomal membrane. Fusion of these two membranes results in the release of the viral RNA into the cytoplasm of the host cell and hence infection. Hemagglutinin is an integral membrane protein which is synthesized as a precursor, HAO, that assembles into a trimer shortly after synthesis. HAO is then proteolytically cleaved to produce two subunits, HA1 and HA2, which remain linked by a single disulfide bridge. HA2 provides an extensively palmitoylated carboxy-terminal membrane anchor and a short cytoplasmic tail. The amino terminus of HA2 is hydrophobic and is required for fusion; it is therefore often referred to as the 'fusion peptide'. The structure of the neutral-pH form of the ectodomain of hemagglutinin, as determined by X-ray crystallography, shows that the fusion peptide is hidden in the stem of the trimer (Fig. la), which consists mainly of HA2 [2]. HA1 makes up most of the membrane-distal domain of the protein and provides the receptor-binding site. There is no fusion-active that low pH involves two
consensus as to the conformation of the form of hemagglutinin at low pH. It seems induces a conformational change which steps: first, the fusion peptide is exposed
Fig. 1. Conformational changes in hemagglutinin. (a) Hemagglutinin trimer at neutral pH (structure of hemagglutinin from Wilson et al. [21, cartoon after Bentz [10]). (b) Fusion-active low pH conformation postulated in [8]. (c) Postulated final fusion-inactive low-pH conformation. A single monomer in (a) is outlined by a thick black line.
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Current Biology 1994, Vol 4 No 6 (Fig. lb), and then the globular heads of the trimer dissociate (Fig. c) (reviewed by White [3]). A very different model for the conformational change has been proposed recently [4] but remains to be confirmed experimentally. The extent to which the globular heads dissociate in the fusion-active conformation is disputed. If two additional cysteines are engineered into HA1, causing monomers of HA1 to cross-link to adjacent monomers via disulfide bridges at the tips of the trimer, exposure of the fusion peptide is impaired and there is no fusion [5,6]. On the other hand, the hemagglutinins from several strains of virus mediate fusion without separation of the globular heads [7]. Those hemagglutinins that do undergo extensive dissociation of the head domains at 37 C can still mediate fusion at 0 C, even though it can be shown, using monoclonal antibodies that recognize epitopes in the tip of the trimer, that head dissociation does not occur at this temperature [8]. When a preparation of virus with these characteristics is taken to low pH at 37°C, in the absence of membranes with which to fuse, the globular heads of hemagglutinin separate. Once the globular heads separate, the molecule quickly becomes inactivated. If virus treated in this way is subsequently added to target membranes, it is no longer able to induce fusion. These results suggest that limited changes at the tip of the trimer are required for fusion, and that a transient intermediate (Fig. lb) represents the fusionactive form of the molecule, whereas the completely dissociated form of hemagglutinin (Fig. c) has no fusion capacity. How could the conformational change in hemagglutinin lead to membrane fusion? Different experimental approaches have led to a plethora of models for hemagglutinin-lipid interactions during fusion. Most workers agree that, after the conformational change, the fusion peptides immediately insert into the target membrane for fusion (although there is not universal agreement on this point; see below). Experiments using photoactivatable lipid analogues in the target membrane have demonstrated that insertion of the fusion peptide precedes the actual merging of the membranes [9]. Furthermore, it is widely believed that fusion is caused by fusion complexes, which are higher order multimers of hemagglutinin trimers, and that fusion is induced by transient intermediate protein-lipid structures. Three of the main models for such intermediates are shown in Figure 2 (in parts a, b and d). The first model (Fig. 2a) [10] differs from most others in not assuming that the fusion peptides insert into the target membrane. Instead, it proposes that the fusion complex consists of a ring of hemagglutinin molecules, with fusion peptides lining the inside of the complex and providing a hydrophobic surface for contact with the lipid tails of an inverted micelle, a sphere or ovoid of lipids that have their hydrophobic tails pointing outward and their hydrophilic heads facing an aqueous interior. Inverted micelles were found to be fusion intermediates in several model systems used for studying
membrane fusion. The micelles would initiate mixing of the lipids from the outer membrane leaflets, then cause rupture of the inner membrane leaflets at the site of micelle formation, resulting in fusion. But it has been shown that hemagglutinin-mediated membrane fusion can take place under conditions in which inverted micelles cannot form [11], and there is no enhancement of fusion under conditions that promote their formation [12]. These findings suggest that the model shown in Figure 2a is probably incorrect. The second model (Fig. 2b) does not make specific assumptions about the conformation of hemagglutinin or the structure of the lipids, but proposes that a proteinaceous 'fusion pore' is formed initially. The model is based on electrophysiological measurements of the fusion of voltage-clamped hemagglutinin-expressing cells with erythrocytes labelled with a fluorescent lipid probe [13]. Upon fusion, a sudden increase in capacitance of the hemagglutinin-expressing cell was recorded, due to the increase in membrane surface area - but the fluorescent lipids diffused into the membrane of the hemagglutinin-expressing cell only much later. This means that an aqueous connection, the 'fusion pore', is formed between the two fusing cells and that it does not initially allow lipid flow. It was therefore proposed that an entirely proteinaceous fusion complex, consisting of a ring of hemagglutinin molecules, would form a water-tight pore through both membranes (Fig. 2b). Following an initial jump, the capacitance was found to 'flicker' a few times; it was therefore suggested [13] that the pore initially opened and closed repeatedly. Subsequently, the pore would widen gradually, somehow acquiring lipids sideways, and initiating fusion. In the third model [3,14,15] (Fig. 2d), the fusion-active form of hemagglutinin would be similar to that shown in Figure lb. At neutral pH, the fusion peptide is 30 A away from the viral membrane but 100 A from the tip of the molecule (Fig. la). Thus, to explain the observed [9,16] insertion of fusion peptides into the target membrane, it is assumed that hemagglutinin must bend (Fig. 2d) [8]. In this model, the fusion peptides in the bent form of the molecule could insert into the viral membrane as well as into the target membrane. Fusioncomplex formation would bring fusion peptides together, and the resulting high local concentration of fusion peptides could create a fault in the target membrane [15]. Beginning at the fault, rotation of the peptides could give rise to the formation of lipidic intermediate structures known as stalks, which are spool-shaped and composed of fused outer membrane leaflets of the viral and target membrane bilayers (Fig. 2d, center) [15]. Rupture of the inner membrane leaflets will then lead to complete fusion giving rise to a lipid-lined fusion pore (Fig. 2d, bottom). The pore may be restrained initially, for example by the inserted fusion peptides, and could revert to a stalk intermediate and back again to a pore repeatedly, which could explain the capacitance flicker mentioned
DISPATCH
Fig. 2. Proposed mechanisms of membrane fusion. (a) Fusion via inverted micelles (after Bentz [10]). (b) One possible interpretation of a proteinaceous fusion pore. (c) Hemifusion (after Kemble et al. [1]). (d) Stalk model for membrane fusion (after Siegel [15] and Stegmann and Helenius [14]): insertion of the fusion peptide into the target and viral membrane; stalk formation; and fusion.
previously [13]. The transmembrane anchor of hemagglutinin could act to lower the activation energy of stalk formation [15] and, if concentrated in the fusion complex, might create a fault in the viral membrane. It has also been suggested that the ectodomain of hemagglutinin cannot bend relative to its membrane anchor: if this is the case, then hemagglutinin would have to tilt as a whole, deforming the viral membrane and creating a bulge that might lower the barriers to membrane fusion [171. To investigate the role of the membrane anchor of hemagglutinin in fusion, Kemble et al. [1] replaced the transmembrane domain of hemagglutinin with a glycosylphosphatidylinositol (GPI) tail, a lipid anchor that inserts into the outer membrane leaflet but not into the inner leaflet. The GPI-tailed hemagglutinin was expressed on the surface of a cell, and its ability to induce. fusion with erythrocytes that contained either water-soluble fluorescent probes or lipid-embedded fluorescent probes was investigated. Surprisingly, the water-soluble probes were not delivered to the cytoplasm of the GPI-hemagglutinin-expressing cell, but transfer of the lipid probes was similar in kinetics and efficiency to that seen with wild-type, non-GPIanchored hemagglutinin. This must mean that the outer
membrane leaflets of the erythrocyte and the GPI-hemagglutinin-expressing cell mixed, but the inner membrane leaflets did not (Fig. 2c), a situation found sporadically in model systems of membrane fusion and termed 'hemifusion'. It therefore appears that hemifusion mediated by GPI-hemagglutinin produces the equivalent of a stalk-like intermediate (see Fig. 2d, center). If GPI-hemagglutinin suffices to induce stalk formation but not full fusion, then the membrane and anchor domains of hemagglutinin must be responsible for rupture and fusion of the inner membrane leaflet of the plasma membrane in which hemagglutinin is embedded [1]. The lipid-lined fusion pore of the stalk model would appear to be incompatible with the results of the electrophysiological measurements, which indicated that the initial pore is proteinaceous because it is formed before the lipids have mixed (Fig. 2b) [13]. But it could be that the ring of hemagglutinin membrane-anchors in the fusion complex acts to prevent, or significantly slow, the flow of lipids through the stalk, in the manner of a tight junction. The GPI tails would not pose such a barrier [1]. As a result of the high curvature of the lipids in a stalk, it is hard to imagine that hemifusion involves
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a stably arrested stalk structure. It is more likely that a dynamic equilibrium between the stage preceding stalk formation (Fig. 2d, top) and stalk structures (Figs 2c and 2d, center) exists. If this is the case, with luck, one may be able to detect fluorescent lipid probes being transferred from an erythrocyte to a GPI-hemagglutinin cell in bursts. And, if stalks are formed all the time, the chances of being able directly to see stalks between the two cells by electron microscopy should be much better. It is also possible that stalks are formed just once, leading to the transfer of lipids, and then the hemagglutinin is inactivated. While these exciting questions can now be addressed experimentally, the results of the GPI-hemagglutinin experiments strongly suggest that fusion involves a stalk-like intermediate, explaining how, at least in this case, a protein gets lipid bilayers to mix. References 1. KEMBLE GW, DANIELI T, WHITE JM: Lipid-anchored influenza 2. 3. 4. 5.
6.
hemagglutinin promotes hemifusion, not complete fusion. Cell 1994, 76:383-391. WILSON IA, SKEHEL JJ, WILEY DC: Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 1981, 289:366-375. WHITE JM: Membrane fusion. Science 1992, 258:917-924. CARR CM, KIM PS: A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell 1993, 73:823-832. GODLEY L, PFEIFER J, STEINHAUER D, ELY B, SHAW G, KAUFMAN R, SUCHANEK E, PABO C, SKEHEL JJ, WILEY DC, WHARTON S: Introduction of intersubunit disulfide bonds in the membranedistal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell 1992, 68:635-645. KEMBLE GW, BODIAN DL, ROSE J, WILSON IA, WHITE JM: Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin. J Virol 1992, 66:4940-4950.
7.
PURI A, BOOY FP, DOMS RW, WHITE JM, BLUMENTHAL R:
Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment. J Virol 1990, 64:3824-3832. 8.
STEGMANN T, WHITE JM, HELENIUS A: Intermediates in influenza
induced membrane fusion. EMBOJ 1990, 9:4231-4241. 9.
STEGMANN T, DELFINO JM, RICHARDS FM, HELENIUS A: The
HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion. J Biol Chem 1991, 266:18404-18410. 10. BENTZ J, ELLENS H, ALFORD D: Architecture of the influenza
hemagglutinin fusion site. In Vtralfusion mechanisms. Edited by Bentz J. Boca Raton: CRC Press; 1993:163-199. 11. STEGMANN T: Influenza hemagglutinin-mediated membrane fusion does not involve inverted phase lipid intermediates. J Bol Chem 1993, 268:1716-1722. 12. ALFORD D, ELLENS H, BENTZ J: Fusion of influenza virus with
sialic acid bearing target membranes. Biochemistry 1994, 33:1977-1987. 13. TSE FW, IWATA A, ALMERS W: Membrane flux through the pore
formed by a fusogenic viral envelope protein during cell fusion. J Cell Biol 1993, 121:543-552. 14. STEGMANN T, HELENIUS A: Influenza virus fusion: from models toward a mechanism. In Viral Fusion Mechanisms. Edited by Bentz J. Boca Raton: CRC press; 1993:89-111. 15. SIEGEL DP: Modeling protein-induced fusion mechanisms: insights from the relative stability of lipidic structures. In Viral Fusion Mechanisms. Edited by Bentz J. Boca Raton: CRC Press; 1993:475-512. 16. TSURUDOME M, GLOCK R, GRAF R, FALCHETTO R, SCHALLER U,
BRUNNER J: Mechanism of influenza induced membrane fusion: membrane insertion of the 'fusion' peptide of hemagglutinin. JBiol Chem 1992, 267:20225-20232. 17. WILSCHUT J, BRON R: The influenza hemagglutinin: membrane fusion activity in intact virions and reconstituted virosomes. In Viralfusion mechanisms. Edited by Bentz J. Boca Raton: CRC Press; 1993:133-161.
Toon Stegmann, Department of Biophysical Chemistry, Biozentrum of the University of Basel, CH 4056, Basel, Switzerland.