By Vivienne Baillie Gerritsen
Bubble formation is not only reserved to the likes of champagne or beer - our own cells also sprout bubbles continuously. These bubbles are more commonly known as vesicles and are formed by means of a much two studied cellular processes: exocytosis and indeed endocytosis. The point of such bubbles in living organisms -plant or animal - is to relieve the plasma membrane of a number of constituents. Whether it be to down regulate a pathway - by dismissing a certain receptor from the plasma membrane for example - or to transport molecules from one side of a cell to another. A certain species of vesicle - the clathrin-coated vesicles - are particularly important in vesicle trafficking in endocytosis as well as exocytosis. And in the case of endocytosis, it is becoming increasingly clear that one protein, epsin, has a major role in the initial steps of membrane budding.
Fig. 1 Adaptor protein, clathrin and epsin in clathrin-mediated vesicle formation.*
A horde of proteins are involved in clathrin-mediated endocytosis: receptors, accessory factors, the clathrin adaptor protein AP2 and of course clathrin. To describe the process rather grossly, specific patches form on the surface of the cell’s membrane; a closer look reveals a clustering of diverse molecules amongst which clathrin which arranges itself into a spheric scaffolding which will form, in effect, the mold into which the membrane will eventually spill. Though spill is not quite the word since the molecules of clathrin lie on the surface of the membrane and accompany the membrane as it moves inwards, thus forming a spherical shell. Without clathrin, there would be no clathrin-coated vesicle. Naturally. What’s more, without epsin, there would be none either. Indeed, one of epsin’s roles is to bind to clathrin - whilst promoting its assembly - as it also binds to the plasma membrane. Yet this is not epsin’s major asset.
Epsin, an accessory protein, was first discovered in 1988 by virtue of its binding to another accessory factor, Eps15, and hence became the Eps15 interacting protein, or epsin for short. The N-terminal domain, otherwise known as the ENTH domain (from epsin NH2 terminal homology domain), is of particular interest. It is a neat, compact bundle of alpha helices, one of which changes its orientation depending on whether epsin is bound to its ligand or not. And what would its ligand be? To cut a long story short, the C-terminal portion of epsin binds to Eps15, its central portion binds both to clathrin adaptor protein AP2 and clathrin itself, and its N-terminal portion - the ENTH domain (from epsin NH2 terminal homology domain) - binds to phosphatidyl inositols. But not any old phosphatidyl inositols: it has a clear preference for phosphatidylinositol-4,5-bisposphates or PtdIns(4,5)P2. This is the ligand upon which epsin will act to initiate cell budding. In what way?
We have all, in our youth, been upset by a balloon which, with a loud bang far too close to our ears, has just burst. However, who hasn’t found some fun in stretching part of the torn remnants of the said balloon between their thumbs to suck out a mini-balloon? This playful rubber invagination is very much what happens in the process of cell endocytosis. Though we were certainly not aware of it, by sucking in a bubble we were applying a certain stress to the rubber to make it bend and curve into a small sphere. Cell endocytosis also demands bending stress to the plasma membrane and the rearrangement of its underlying structure, first to form a budding vesicle and then the vesicle in its entirety. Well, it seems that epsin has found a way to rearrange the underlying structure of the plasma membrane so that it curves spontaneously. How?
When epsin spots a patch of PtdIns(4,5)P2 within a lipid layer, it inserts part of its N-terminal ENTH domain into the plasma membrane. It does this by way of a short alpha helix. This helix is rather special since, though epsin is quite a soluble protein, it is literally inside out, so to speak, because it presents hydrophobic residues on its outer surface. When this amphipathic helix encounters a membrane enriched in PtdIns(4,5)P2, it folds back onto the other neatly bundled helices and forms a pocket into which the PtdIns(4,5)P2 head can fit. All this movement disrupts the organization of the lipid layer causing it to find another arrangement. The effect is not dissimilar to that caused by a rather large man who, despite a tight squeeze, decides to seat himself on a bench, thus making all those already seated to shift a little on either side of him to make space. This shift in location offers a perfect opportunity for the membrane to curve as the lipids rearrange in the membrane. Why? It is all a question of expansion, contraction, tension, surface pressure, energy imbalance… and could well be explained by the fact that epsin insertion into the lipid layer creates chemical asymmetry in the plasma membrane as a whole, i.e. a difference arises between the interfacial tension and the internal surface pressure of the membrane, the result of which is the curving of the membrane.
So not only does epsin stimulate clathrin assembly and may well act as an initial scaffolding onto which the clathrin cage could grow, but it also causes the membrane to camber, thanks to its ENTH domain. In fact, the popularity of the ENTH domain is growing fast. Indeed, it does show hints of recruiting and bridging vesicle coat components other than clathrin and could well have a role not only in forming the container in which cargo is transported but also in transporting the cargo itself.
* Source: http://www2.mrc-lmb.cam.ac.uk/groups/hmm/epsin/EM/MonolayerEMs.html
Cross-references to Swiss-Prot
Q9Y6I3: human epsin 1
O88339: rat (Rattus norvegicus) epsin 1
References
1. Hurley J.H., Wendland B.
Endocytosis: driving membranes around the bend
Cell 111:143-146(2002)
PMID: 12408856.
2. Ford M.G.J., Mills I.G., Peter B.J., Vallis Y., Praefcke G.J.K., Evans P.R., McMahon H.T.
Curvature of clathrin-coated pits driven by epsin
Nature 419:361-366(2002)
PMID: 12353027.
3. Nossal R., Zimmerberg J.
Endocytosis: curvature to the ENTH degree
Curr. Biol. 12:770-772(2002)
PMID: 12445401.
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