Nsf

a-SNAP

c/s-SNARE /^t complex

Disassembly of SNARE complexes

from syntaxin, and two from SNAP-25—to coil around one another to form a four-helix bundle. The unusual stability of this bundled SNARE complex is conferred by the arrangement of hydrophobic and charged amino residues in the heptad repeats. The hydrophobic amino acids are buried in the central core of the bundle, and amino acids of opposite charge are aligned to form favorable electrostatic interactions between helices. As the four-helix bundles form, the vesicle and target membranes are drawn into close apposition by the embedded transmembrane domains of VAMP and syntaxin.

In vitro experiments have shown that when liposomes containing purified VAMP are incubated with other liposomes containing syntaxin and SNAP-25, the two classes of membranes fuse, albeit slowly. This finding is strong evidence that the close apposition of membranes resulting from formation of SNARE complexes is sufficient to bring about membrane fusion. Fusion of a vesicle and target membrane occurs much more rapidly and efficiently in the cell than it does in liposome experiments in which fusion is catalyzed only by SNARE proteins. The likely explanation for this difference is that in the cell the interactions between specific Rab proteins and their effectors promote the formation of specific SNARE bundles by tethering a vesicle to its target membrane.

Yeast cells, like all eukaryotic cells, express more than 20 different related v-SNARE and t-SNARE proteins. Analyses of yeast sec mutants defective in each of the SNARE genes have identified the specific membrane-fusion event in which each SNARE protein participates. For all fusion events that have been examined, the SNAREs form four-helix bundled complexes, similar to the VAMP/syntaxin/SNAP-25 complexes that mediate fusion of secretory vesicles with the plasma membrane. However, in other fusion events (e.g., fusion of COPII vesicles with the cis-Golgi network), each participating SNARE protein contributes only one a helix to the bundle (unlike SNAP-25, which contributes two helices); in these cases the SNARE complexes comprise one v-SNARE and three t-SNARE molecules.

Using the in vitro liposome fusion assay, researchers have tested the ability of various combinations of individual v-SNARE and t-SNARE proteins to mediate fusion of donor and target membranes. Of the very large number of different combinations tested, only a small number mediated membrane fusion. To a remarkable degree the functional combinations of v-SNAREs and t-SNAREs revealed in these in vitro experiments correspond to the actual SNARE protein interactions that mediate known membrane-fusion events in the yeast cell. Thus the specificity of the interaction between SNARE proteins can account for the specificity of fusion between a particular vesicle and its target membranes.

Dissociation of SNARE Complexes After Membrane Fusion Is Driven by ATP Hydrolysis

After a vesicle and its target membrane have fused, the SNARE complexes must dissociate to make the individual SNARE proteins available for additional fusion events. Be cause of the stability of SNARE complexes, which are held together by numerous noncovalent intermolecular interactions, their dissociation depends on additional proteins and the input of energy.

The first clue that dissociation of SNARE complexes required the assistance of other proteins came from in vitro transport reactions depleted of certain cytosolic proteins. The observed accumulation of vesicles in these reactions indicated that vesicles could form but were unable to fuse with a target membrane. Eventually two proteins, designated NSF and a-SNAP, were found to be required for ongoing vesicle fusion in the in vitro transport reaction. The function of NSF in vivo can be blocked selectively by N-ethylmaleimide (NEM), a chemical that reacts with an essential -SH group on NSF (hence the name, NEM-sensitive factor).

Among the class C yeast sec mutants are strains that lack functional Sec18 or Sec17, the yeast counterparts of mammalian NSF and a-SNAP, respectively. When these class C mutants are placed at the nonpermissive temperature, they accumulate ER-to-Golgi transport vesicles; when the cells are shifted to the lower, permissive temperature, the accumulated vesicles are able to fuse with the cis-Golgi.

Subsequent to the initial biochemical and genetic studies identifying NSF and a-SNAP, more sophisticated in vitro transport assays were developed. Using these newer assays, researchers have shown that NSF and a-SNAP proteins are not necessary for actual membrane fusion, but rather are required for regeneration of free SNARE proteins. NSF, a hexamer of identical subunits, associates with a SNARE complex with the aid a-SNAP (soluble NSF attachment protein). The bound NSF then hydrolyzes ATP, releasing sufficient energy to dissociate the SNARE complex (Figure 17-11, step 4). Evidently, the defects in vesicle fusion observed in the earlier in vitro fusion assays and in the yeast mutants after a loss of Sec17 or Sec18 were a consequence of free SNARE proteins rapidly becoming sequestered in undissoci-ated SNARE complexes and thus unavailable to mediate membrane fusion.

Conformational Changes in Viral Envelope Proteins Trigger Membrane Fusion

Some animal viruses, including influenza virus, rabies virus, and human immunodeficiency virus (HIV), have an outer phospholipid bilayer membrane, or envelope, surrounding the core of the virus particle composed of viral proteins and genetic material. The viral envelope is derived by budding from the host-cell plasma membrane, which contains virus-encoded glycoproteins. Enveloped viruses enter a host cell by endocytosis following binding of one or more viral envelope glycoproteins with a host's cell-surface molecules. Subsequent fusion of the viral envelope with the endosomal membrane releases the viral genome into the cytosol of the host cell, initiating replication of the virus (see Figure 4-41, step 3). The molecular events of this fusion process have been elucidated in considerable detail in the case of influenza virus.

Cell-surface membrane

Fusion peptide

Cell-surface membrane

Fusion peptide

Disulfide bond

Viral envelope

Disulfide bond

Viral envelope

Endosomal membrane

Endosomal membrane

▲ FIGURE 17-12 Schematic models of the structure of influenza hemagglutinin (HA) at pH 7 and 5. Three HA1 and three HA2 subunits compose a hemagglutinin molecule, which protrudes from the viral envelope like a spike. (a) At pH ~7, part of each HA1 subunit forms a globular domain (green) at the tip of the native spike. These domains bind to sialic acid residues on the host-cell plasma membrane, initiating viral entry. Each HAt subunit is linked to one HA2 subunit by a disulfide bond at the base of the molecule near the viral envelope. Each HA2 subunit contains a fusion peptide (red) at its N-terminus (only two are visible), followed by a short a helix (orange cylinder), a nonhelical loop (brown), and a longer a helix (light purple). The longer a helices from the three HA2 subunits form a three-stranded coiled-coil structure (see Figure 3-7). In this conformation, the fusion peptides are buried within the molecule. (b) At the acidic pH within a late endosome, the binding of the fusion peptide to other segments of HA2 is disrupted, inducing major structural rearrangements in the protein. First, the three HAt globular domains separate from each other but remain tethered to the HA2 subunits by the disulfide bonds at the base of the molecule. Second, the loop segment of each HA2 rearranges into an a helix (brown) and combines with the short and long a-helical segments to form a continuous 88-residue a helix. The three long a helices thus form a 13.5-nm-long three-stranded coiled coil that protrudes outward from the viral envelope. In this conformation, the fusion peptides are at the tip of the coiled coil and can insert into the endosomal membrane. [Adapted from C. M. Carr et al., 1997, Proc. Nat'l. Acad. Sci. 94:14306; courtesy of Peter Kim.]

The predominant glycoprotein of the influenza virus is hemagglutinin (HA), which forms the larger spikes on the surface of the virus. There is considerable evidence that following endocytosis of an influenza virion, the low pH within the enclosing late endosome triggers fusion of its membrane with the viral envelope. For instance, viral infection is inhibited by the addition of lipid-soluble bases, such as ammonia or trimethyl-amine, which raise the normally acidic pH of late endosomes. Also, a conformational change in the HA protein that is critical for infectivity occurs over a very narrow range in pH (5.0-5.5).

Each HA spike on an influenza virion consists of three HA1 and three HA2 subunits. At the N-terminus of HA2 is a strongly hydrophobic 11-residue sequence, called the fusion peptide. Structural studies have shown that at pH 7.0, the N-terminus of each HA2 subunit is tucked into a crevice in the spike (Figure 17-12a). This is the normal HA conformation when a viral particle encounters the surface of a host cell. At the acidic pH characteristic of late endosomes, HA undergoes several conformational changes that cause a major rearrangement of the subunits. As a result, the three HA2 subunits twist together into a three-stranded coiled-coil rod that protrudes more than 13 nm outward from the viral envelope with the fusion peptides at the tip of the rod (Figure 17-12b). In this conformation, the highly hydrophobic fusion peptides are exposed and can insert into the lipid bilayer of the endosomal membrane, triggering fusion of the viral envelope and the membrane. Thus at pH 7 HA can be said to be trapped in a metastable, "spring-loaded" state, which is converted to the lower-energy fusogenic state by shifting the pH to 5-5.5.

Multiple low pH-activated HA spikes are essential for membrane fusion to occur. Figure 17-13 suggests one way by which the protein scaffold formed by many HA spikes, possibly with the assistance of other cellular proteins, could link together the

Endosomal membrane

Activated HA proteins

Endosomal membrane

Activated HA proteins

/Viral envelope

/Viral envelope

Cystolic leaflet

Cystolic leaflet

Exoplasmic leaflet

Fused membranes

Exoplasmic leaflet

Fused membranes

▲ FIGURE 17-13 Model for membrane fusion directed by hemagglutinin (HA). A number of low pH-activated HA spikes, possibly in concert with host-cell membrane proteins, form a scaffold that connects a small region of the viral envelope and the endosomal membrane. By unknown mechanisms, the exoplasmic leaflets of the two membranes fuse and then the cytosolic leaflets fuse, forming a pore that widens until the two membranes are completely joined. A similar interaction between membrane bilayers may be brought about during SNARE-mediated vesicle fusion. [Adapted from J. R. Monck and J. M. Fernandez, 1992, J. Cell Biol. 119:1395.]

viral envelope and endosomal membrane and induce their fusion. This figure also illustrates how cellular membranes brought into close apposition by SNARE complexes might fuse. Note that each HA molecule participates in only one fusion event, whereas the cellular fusion proteins, such as SNAREs, are recycled and catalyze multiple cycles of membrane fusion.

and some of the evidence supporting the general mechanisms discussed in the previous section. Recall that anterograde transport from the ER to Golgi, the first step in the secretory pathway, is mediated by COPII vesicles, whereas the reverse retrograde transport from the cis-Golgi to the ER is mediated by COPI vesicles (Figure 17-14). This retrograde

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