Lipids and membrane fusion

Membrane fusion is a very common process in cells. In eukaryotic organisms, many different fusion events can occur at any given time, participating in such diverse functions as secretion, endocytosis, intracellular digestion in lysosomes, cell division and the adjustment of mitochondrial numbers to a cell's energy requirements. Just a short consideration of the overall events of fusion will make it obvious that, during the process, the normal lipid bilayer structure must disappear.

First of all, biochemists studied the process in model systems, employing lipid vesicles. It has been recognized for some time that such vesicles can be induced to fuse when incubated in the region of their gel to liquid-crystalline transition temperature. Furthermore, it was also noted that Ca2+ addition would induce fusion in phosphati-dylserine-containing systems, possibly by inducing phase separation of the lipid and, hence, a local crystalline point for fusion. However, it was also noted that in more complex mixtures, Ca2+ was not always able to induce fusion.

An important clue as to the mechanism of lipid membrane fusion has come from the work of de Kruijff and Cullis. They proposed that fusion proceeded because of the ability of lipids to undergo polymorphism (i.e. to adopt different structures). Three types of observation support the hypothesis. First, fusogens (such as monoacylglycerols) induce HII (inverted micellar structures; Fig. 6.12) phase structures, consistent with a role for non-bilayer structures in fusion. Second, promotion of fusion of, for example, phosphatidylserine-containing systems by Ca2+ is accompanied by HII structures. Third, a number of factors, such as pH changes or elevated temperatures that cause HII formation, also promotes fusion of lipid vesicles.

Siegel and coworkers have examined fusion in more detail particularly in relation to two possible mechanisms - via inverted micelle intermediates or by stalk structures (Fig. 6.18). They produced evidence for either process being important although more recent experiments favour the stalk hypothesis. In either mechanism, however, non-lamellar lipid phases are initially involved. Furthermore, artificial membranes with inverted cone-shaped lipids (Fig. 6.12, supporting positive curvature) added to the cis-sided leaflets block stalk formation whereas their addition to trans-leaflets promotes pore opening. Cone-shaped lipids (Fig. 6.12) have the opposite effects. These results illustrate the potential for different-shaped lipids to influence membrane stability in various structures (Section 6.5.11).

Cone Shaped Lipid
Fig. 6.18 Two possible mechanisms for membrane fusion. In method (a) inverted micellar intermediates (IMI) are involved and, in (b), the formation of a stalk. Reproduced from Cullis et al. (1996) with kind permission of the authors and Elsevier Science.

Extension of the Cullis-de Kruijff hypothesis to natural membrane events has been difficult to prove. Nevertheless, confirmatory evidence has been obtained with a number of systems such as the release of chromatin granule contents during stimulation of the adrenal medulla. The exocytosis accompanying this event seems to depend on the ability of Ca2+ to promote non-bilayer structures.

Evidence from freeze-fracture microscopy has also implicated hexagonal II phases in two natural membrane systems where a kind of 'arrested fusion7 exists. These are in the tight junctions between epithelial and endothelial cells and in the contact sites between the inner and outer membranes of mitochondria or of Gram-negative bacteria like E. coli.

Fusion of biological membranes is now thought to require the action of specific fusion proteins. Of these, the viral fusion proteins that help the virus fuse with the host cell membrane during infection are the best understood. These proteins undergo dramatic and spontaneous conformational changes upon activation. For the influenza and the human immunodeficiency (HIV) viruses, the fusion pep-tide inserts itself into the membrane and then re orients itself thus forcing the fusion membranes together and allowing lipid mixing.

Fusion of intracellular eukaryotic membranes involves several protein families. These are termed SNAREs [soluble NSF (N-ethylmaleimide Sensitive Factor) receptors], SM proteins (Sec 1/Munc 18 homologues where, for example, Sec derives from sec mutants of yeast that have temperature-sensitive blocks in the secretory pathway) and Rab proteins (small GTPases, which in yeasts genetically interact with SNAREs and SM proteins). The latter probably function in the initial membrane contact connecting the fusion proteins, but are not involved in fusion itself. Although there is much genetic and structural information about these three components, which are essential for fusion (evidence from gene deletions or mutations), exactly what they do in the process is still unclear. Furthermore, although the mechanism of membrane fusion seems broadly similar in different situations, there must clearly be specificity to proteins such as in intracellular membrane traffic.

In the foregoing section we have suggested ways in which cells could initiate membrane fusion through allowing areas of hexagonal II phase.

Conversely, there may be situations where the synthesis of large amounts of hexagonal II-forming lipids could destabilize the normal bilayer structure. Thus, when the anaerobic bacterium Clostridium butyricum is grown in the absence of biotin (when it is reliant on exogenous fatty acids) its membrane lipid composition can be made more unsaturated by supplying cis-monounsaturated acids rather than saturated ones. Since phosphati-dylethanolamine and its plasmalogen are major membrane lipids in the bacterium, and their unsa-turated species form the hexagonal II phase (Fig. 6.12) an increase in unsaturation might destabilize the membrane. C. butyricum reacts by reducing the proportion of phosphatidylethanolamine and increasing that of its glycerol acetal. Since the latter forms bilayers then this change restabilizes the membrane structure. Similarly, in Acholeplasma laid-lawii where monoglucosyldiacylglycerol and diglucosyldiacylglycerol are major membrane lipids, the former is hexagonal II-forming while the latter gives bilayers (Fig. 6.12). Under conditions such as increased temperature or unsaturation, which would tend to favour the formation of non-bilayer structures, the monoglucosyl-lipid is converted to diglucosyldiacylglycerol. This change in lipid proportions would again be expected to preserve membrane bilayer stability.

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