Membrane fusion, in which two distinct lipid bilayer membranes are merged into one, is the common final step in the transport of proteins among intracellular compartments, the controlled release of hormones and neurotransmitters by exocytosis, and the penetration of the genomes of enveloped viruses into the cytoplasm. Remarkably, common physical principles appear to underlie these diverse biological processes that involve fusion on both sides of cellular membranes.
Viral membrane fusion is mediated by extracellular viral-encoded proteins embedded in the viral envelope that are activated by receptor binding at the cell surface or endocytosis (Dalgleish et al. 1984
; Klatzmann et al. 1984
; Maddon et al. 1986
; Feng et al. 1996
). Upon activation, they can undergo a dramatic conformational switch to form a helical bundle; like the SNAREpin, this viral hairpin is simultaneously inserted into both membrane partners (reviewed by Skehel and Wiley 1998
). Before activation, viral fusion proteins are anchored solely in the viral envelope. Therefore, during this switch, the viral protein must insert into the cellular membrane. This is typically mediated by a specialized fusion peptide that is buried in the protein structure before activation (Wilson et al. 1981
These models raise the fundamental question of whether membrane fusion is an essentially passive or an active process. It could be that simply pinning two bilayers together long enough is sufficient for fusion; the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning, in one or another way perturbing the lipids by exerting force in the bilayer. The assembly of the helical bundles that comprise pins would seem to afford an ample source of energy to do work on the bilayer, since isolated SNARE complexes and viral helical bundles only denature at temperatures above 80–90°C (Hayashi et al. 1994
; Lu et al. 1995
; Lamb et al. 1999
In principle, it is possible to distinguish an active from a passive mechanism by examining the effect upon fusion activity of replacing one or both of the peptidic membrane anchors of fusogenic pins with covalently attached lipids. If fusion occurs passively, it should not be adversely affected by lipid anchors since simply holding the bilayers together long enough is by definition sufficient. If, however, fusion is found not to occur with some or all species of lipid anchors, then it necessarily involves an active mechanism, since in at least some cases simply holding the bilayers together long enough did not suffice. Of course, for these conclusions to hold, the separation of the two bilayers must remain constant independent of the nature of the anchor, or equivalently, fusion must be shown to be insensitive to variations in separation in the range resulting from substituting lipid anchors. Also, the assembly of the pin must be unaffected by lipid substitution.
Elegant and pioneering studies have shown that replacing the natural peptidic anchor that holds viral fusion proteins into the envelope with an encodable phospholipid-based glycerophosphoinositide (GPI) unit prevents fusion and results in hemifusion, a state where outer leaflets merge, but the inner leaflets do not (Kemble et al. 1994
; Melikyan et al. 1995
). However, a recent study suggests that GPI-linked HA can produce nonexpanding fusion pores under certain experimental conditions (Markosyan et al. 2000
). These results establish the importance of the viral membrane anchor and are consistent with the possibility that fusion involves an active mechanism. However, it is also possible that hemifusion and subsequent effects are a unique consequence of a GPI-anchor and the viral fusion peptide. Insertion of the fusion peptide may destabilize bilayer structure and possibly facilitate the bilayer-to-nonbilayer transition that is likely required for hemifusion. Unfortunately, it is not presently possible to lipid-anchor viral fusion proteins in both the viral and target membrane to eliminate viral fusion peptide-specific phenomena.
By contrast, SNARE proteins are well suited for such studies because the two membrane anchors can be manipulated in separate polypeptide chains and tested later when reconstituted into liposomes. Here, we make precisely such changes in cognate SNAREs mediating exocytosis, vesicle-associated membrane protein 2 (VAMP2, the v-SNARE), a heterodimer of syntaxin1A, and a synaptosomal-associated protein of 25 kD (SNAP-25B, the t-SNARE; Trimble et al. 1988
; Baumert et al. 1989
; Oyler et al. 1989
; Bennett et al. 1992
; Sollner et al. 1993
). We find that close proximity of two membranes is not sufficient for fusion, though there are certain lipid anchors that appear to permit lipid mixing. Additionally, we describe the synthesis and utilization of four novel isoprenoid lipid anchors.