Membrane fusion

 

Membrane fusion is the process in which two opposing membranes are joined into one contiguous membrane.

In synthetic systems, i.e. liposomes membrane fusion is readily achieved upon adding divalent cations such as Ca2+ to liposomes containing negatively charged lipids.

There are major differences between this sort of membrane fusion and that found in biological systems in that in biological system:

• The proteins that reside in the membrane retain their relative orientations in biological fusion. In ``synthetic'' fusion events proteins tend to get ``randomised''.
• There is no ``leakage'' of ions or any other small compounds in biological fusion. In synthetic fusion events leakage does take place.

It is therefore not surprising that biological membrane fusion involves the action of proteins to maintain and uphold these stringent requirement.

Membrane fusion events can be classified into 2 categories:

* exoplasmic fusion

* endoplasmic fusion

The difference between endoplasmic and exoplasmic fusion relates to the nature of the interacting bilayer leaflets.

Interestingly the mechanism of fusion seems to be dramatically different between these two processes and requires completely different sets of proteins.

Exoplasmic membrane fusion

 

Exoplasmic membrane fusion takes place when the interacting leaflets of the bilayer are those facing the extracellular space, or any other space in the cell separated by an even number membranes (such as the ER and Golgi lumen).

Example of such fusion events are several:

• Zygote formation, i.e. fusion of the sperm and the oocyte.
• Fusion of a virus and a cell membrane
• Cardiac syncytium formation upon fusion of several sarcomers to form a continuos cytoplasm.

 

 

The machinery involved in this sort of fusion event seems to be a single spike protein residing on the membrane of one of the opposing membranes .

 

Proteins such as these have been found on the sperm plasma membrane as well as in the virus envelope. The best characterised of these fusogenic proteins is the influenza trimeric hemagglutinin spike glycoprotein (or HA).

This protein is made up of a single chain that is then cleaved into two chains held together by disulphide bonds. Cleavage results in the ``release'' of the amino-terminus of the b chain, that contains a stretch of hydrophobic amino-acids. This is considered to be the fusion peptide, i.e. the part of the protein that inserts into the target membrane to initiate membrane fusion.

(Note that if the protein were not cleaved, this segment would only be part of the contiguous chain of the proteins and not able to do its job.)

 

Membrane fusion is initiated by acidification that triggers a conformation change in the proteins.

• In the neutral pH form, the fusion peptide is ``hidden'' in the interior of the protein, while in the acid form it is exposed.
• The reason for the exposure is that a stretch of residues (40-100) in the neutral form of the protein form a helix followed by a random coil followed by another helix (helix-turn-helix).
• Exposure to acidic conditions ``converts'' the stretch of residues into one long helix.

 

Fusion of a virus and a cell membrane

Other viruses contain spike glycoproteins that share common features to HA such as cleavage generating a hydrophobic N-terminus of the ``B'' chain.

Most of them are also trimeric such as HA.

Perhaps the most famous example is the GP120 spike glycoprotein from HIV.

• Note that in exoplasmic membrane fusion, the two membranes are not equivalent in terms of their ``desire'' to undergo fusion.
• The ``interested membrane'' (such as the virus envelope) contains all of the machinery required for fusion.
• In fact in laboratory experiments fusion of viruses to liposome can readily be achieved upon acidification.
• This situation is drastically distinct from endoplasmic membrane fusion.

 

Exoplasmic membrane fusion specificity

 

The specificity in the interaction, that is making sure that the correct membranes fuse is a crucial component of the fusion machinery.

One can imagine that none of us would be sitting here if the sperms would fuse with one another sperm instead of the oocyte.

The same is true with viruses targeting a host membrane.

 

 

So how then does specificity arise?

 

• It arises from proteins on the membrane interested in the fusion event, recognising particular attributes on the opposing membranes.
• In Influenza virus for example, the same spike glycoprotein the initiates fusion (HA) first binds to sialic acid (an N-Acyl derivative of neuraminic acid) moieties located on the host cell membrane.
• Interestingly the virus contains an enzyme (neuraminidase) that removes this sugars so that ``over infection'' does not take place.

 

Endoplasmic membrane fusion mechanism & specificity

 

Endoplasmic membrane fusion occurs in all ``communication'' events between different cellular compartments, such as vesicles that have bud from ER fusing with the Golgi, chromafin granules fusing with the plasma membrane, synaptic vesicles full of neurotransmitters fusing with the plasma membrane, etc.

 

 

The mechanism behind these fusion events seems to be dramatically different from exoplasmic fusion and involve a multitude of components utilising ATP.

 

The mechanism of endoplasmic membrane fusion is not that well understood.

The common theme behind endoplasmic membrane fusion is the SNARE Hypothesis. The SNARE hypothesis delineates between the target membrane and the vesicle membrane that partake in the fusion reaction. The vesicle membrane contains an element that specifies its destination (a V-SNARE), while the target membrane contains elements that designates its identity (a T-SNARE).

Interests in the SNARE hypothesis began at an improbable biological juncture between yeast and neurones. Yeast has long been used as a genetic tool in biological research and has been especially useful in the field of vesicular transport. Many genetic mutations and subsequent proteins have been identified that result in defective and blockage in particular stages in vesicular transport. Considerable research in the last several years have identified numerous proteins that are critical in synaptic vesicle maturation and fusion. Interestingly many of these proteins are homologous to yeast proteins and it was shown that vesicular transport in both these eukaryotic systems is very similar.

Since then many more V-SNAREs and T-SNAREs have been discovered in a variety of tissues, and in fact, it is assumed that most if not all intracellular vesicular transport utilises the SNARE machinery.

 

Why the irritating names: SNARE.

 

Well this all has to do with historical reasons.

• Both in-vivo and in-vitro experiments were able to show that adding N-ethylmaleimide(an agent that blocks the sulphydryl groups in cysteine residues commonly called NEM) abolishes endoplasmic membrane fusion. A protein was later identified as the target for this chemical and subsequently called NSF, NEM sensitive fusion protein (note that NSF is also the acronym for the US national science foundation). NSF is a soluble protein, and would therefore require additional factors in order to elicit fusion.
• Additional soluble factors were identified that bind NSF and were therefore called SNAP for soluble NSF attachment proteins.
• Later on membrane proteins were identified that were able to bind NSF and SNAPs and were therefore called soluble NSF attachment protein receptors.
• These SNAREs were then divided according to their location to V-SNAREs are located on vesicles while T-SNAREs are located on the target membrane.

 

So how is fusion supposed to take place?

 

• First the V-SNARE and the T-SNARE bind each other, resulting in juxtaposing the target and vesicle membranes. Recently a structure for a neuronal V-SNARE and the T-SNARE complex was solved by x-ray crystallography.

 

• In a second stage SNAPs and NSF join the V-SNARE-T-SNARE complex and form a large 20S ``fusion particle''.

 

• ATP is hydrolysed and membrane fusion takes place and the complex dissociated

All this sounds very nice however recently ``troublesome'' finding have plagued the SNARE hypothesis.

Perhaps the worst detriment in the rosy world of the SNARE hypothesis is the fact V-SNAREs T-SNAREs may be found in the same membrane

 

When things go wrong, or not at all in endoplasmic membrane fusion

 

Some of the most lethal toxins secreted by clostridia bacteria target elements of the SNARE complex directly.

The toxins are usually composed of 2 polypeptides chains:

• one of which binds to elements in the neuronal tissue and helps it to internalise,
• while the second polypeptide is an incredibly specific protease that cleaves one of the components of the SNARE complex.

 

 

Toxin SNARE Target Cleavage site Cell type Tetanus synaptobrevin a V-SNARE Q76-F77 neurone Tetanus cellubrevin a V-SNARE ? all cells botulinum neurotoxin A SNAP2 a SNAP near C terminus neurone botulinum neurotoxin B synaptobrevin a V-SNARE Q76-F77 neurone botulinum neurotoxin C syntaxin a t-SNARE near C terminus neurone botulinum neurotoxin D synaptobrevin V-SNARE K59-L60 neurone botulinum neurotoxin E SNAP25 a V-SNARE ? neurone botulinum neurotoxin F synaptobrevin V-SNARE Q58-K59 neurone botulinum neurotoxin F cellubrevin a V-SNARE ? all cells

 

Membrane fission & Endocytosis

 

Membrane fission events are extremely common events in cellular biology.

Most, if not all vesicles start their ``life'' as a result of a membrane undergoing budding or invagination.

Receptor mediated endocytosis:

This process is responsible for the transport of specific proteins (e.g. growth factors, iron binding proteins, antibodies) into the cell.

Transport involves initial binding of the proteins to transmembrane protein receptors on the outer surface of the plasma membrane followed by invagination of portions of the membrane to form vesicles containing the protein-receptor complex.

Endocytosis begins when proteins bound to receptor accumulate in ``coated pits'' - specialised regions of the membrane where it is indented and coated on its cytoplasmic side with a bristle-like coat composed of two proteins: clathrin and protein adapters.

 

• Each clathrin triskelion consists of three clathrin heavy chains (180kDa) and three clathrin light chains (35kDa). This clathrin unit has a typical three legged structure in the electron microscope, hence the name triskelion.
• A number of triskelions assemble into a lattice-like coat or clathrin basket on the cytoplasmic surface of the coated pit.
• The inner shell of the basket is composed of the adapter proteins which interact both with the cytoplasmic domains of the receptors and with clathrin.

Rapid internalisation of the receptors requires the tetrapeptide sequence Asn-Pro-X-Tyr which occurs in the first 22 residues of the receptor cytoplasmic domain. Following the assembly of the coated pit, it is then excised or pinched off from the membrane to form a coated vesicle.

Measurements suggest that 2,500 coated vesicles leave the inner surface of the plasma membrane every minute.

 

Very quickly after coated vesicles leave the plasma membrane, an uncoating ATPase removes the clathrin ``overcoats'' from the vesicle which then fuse with cytoplasmic vesicles termed endosomes.

The internal pH of the endosome is maintained between pH 5 & 6 by ATP-driven proton pumps.

The effect of this low pH within the endosome is generally to cause dissociation of the ligand from the receptor.

Often but not always, the ligand passes to another cytoplasmic vesicle, the lysosome to be degraded by proteases.

The receptor recycles back to the cell surface to bind another ligand molecule.

The whole cycle may take only 10 minutes.

 

Viral uptake

 

Some viruses make use of most of the cellular machinery in order to infect the cell and destroy it.

Influenza A is a member of the orthomyxoviruses, which are enveloped viruses containing RNA as their genetic material. Influenza is responsible for some of the most devastating plagues in world history such as the pandemics of 1743, 1889-1890 and 1918-1919, the later resulting in the death of approximately 40-100 million people.

 

• The 1st stage in virus infection is binding of the virus hemagglutinin spike glycoprotein to sialic acid moieties on the host cell.
• The virus then get endocytosed (internalised) by the host cell as explained above.
• The endocytic vesicle containing the virus acidifies due to the function of the resident H+ pump, which in turn results in a conformational change of the hemagglutinin protein. This conformational change causes the fusion peptide to extrude from the proteins which results in fusion of the viral membrane with the endocytic vesicle.
• After fusion has taken place the viral nucleic acid is released and more virus products are made by the host cell.
• The viral proteins are transported to the membrane plasma membrane following the ``standard'' pathway that cellular proteins undertake (ER Golgi plasma membrane).
• The virus then assembles on the plasma membrane through interaction between the spike glycoproteins and the nucleic acid, followed by budding of mature viruses.

 

One of the things that puzzled scientists for some time was that fact that pH of vesicles along the Golgi secretory pathway is acidic enough to cause the conformation induced fusion of the hemagglutinin protein. Why then does fusion not take place when the HA is exported to the plasma membrane?

It does not because the virus makes a small ion channel (that does not get included in the final virion for obvious reasons) that uncouples the resident H+ pump, thereby allowing the protein to remain in its pre-fusion capable form.