Discuss How The Specificity Is Achieved In Membrane Trafficking Pathways

The specificity of vesicle fusion is achieved in three ways 1) vesicle transport, 2) tethering of vesicles to target membranes, and 3) membrane fusion. Protein-protein interaction enables these three stages to only be possible for a small subset of vesicles and membranes ensuring that the fusion of a vesicle and an incorrect membrane is not possible. In this essay I will

In vesicle transport, a vesicle is associated with the cytoskeleton and a molecular motor protein, for example microtubules and kinesin. The direction of transport can be controlled by the type of motor protein attached to the vesicle. For example, kinesin moves towards the + end of MTs and dynein moves towards the - end of MTs. The organisation of microtubules or actin filaments, can act as a control of specificity of vesicles as it will direct the cargo in the right direction and to the desired location. This may be the plasma membrane of the cell or the Golgi membrane or an organelle such as mitochondira. This level of specificity has it's limits though as a vesicle could encounter an organelle in transit to the desired location or may have to pass through an organelle which the cytoskeleton and motor proteins have no control over.

A higher level of specificity is achieved by the tethering of vesicles. There are two types of tethering proteins, coiled coil structures and large protein complexes. Coiled coil tethers are almost always dimers and can be observed within cells under microscopy as thread seeming to link a vesicle to the Golgi or the membrane. Tethering factors bind simultaneously to the cargo and to the target membrane and, as there are only specific combinations of vesicle and membrane that allow the tethering protein to bind, there is specificity. The factors are pathway specific, for example the TRAPPI only functions in the ER. Tethering factors interact with Rab proteins to ensure the correct vesicle docks at the correct site on the correct membrane. Rab proteins are also specific, for example Rab1 is found on the ER. They are molecular switches that are active in their GTP bound form in which they anchor themselves to their target membrane. In this form they can bind Rab effectors such as tethering factors. These tethering factors can then only bind to certain vesicles and allow fusion. Tethering factor p115 binds COP1 vesicles to the Golgi. The tethering factors allow fusion by two possible mechanisms- kinetic mechanism of simply holding the vesicle close to the SNARE or thermodynamic where the tethering promotes SNARE fusion. Coiled coil could be former and multimeric complex could be latter with the ability to transduce the signal to the SNARE.

Another level of specificity occurs at the actual fusion of the vesicle and the membrane, which is achieved by SNARE proteins. These are integral membrane proteins that are found in both the vesicle (v-SNARE), a synaptobrevin-like molecule, and the target membrane (t-SNARE), a syntaxin-like molecule. All SNAREs have a conserved helical domain called a SNARE domain that consists of 60-70 amino acids. These domains can interact with each other by forming coiled coil structures called trans-SNAREs and this process releases energy that can drive membrane fusion by overcoming the repulsion of the membranes. The structure is composed of four helices, one syntaxin 1 and two SNAP-25 found in the target membrane and one synaptobrevin found in the vesicle membrane. The centre of the bindle of 4 a-helices contains 16 layers of interacting side chains, which are highly hydrophobic bar the central layer 0 that contains conserved arginine and glutamine residues. The 'zipper' hypothesis states that the fusion of the SNAREs starts at the N terminus of the proteins and moves towards the C terminus that in anchored in the membrane.

Specificity can be achieved in several ways. Firstly the topological isolation of a v-SNARE and a t-SNARE. The t-SNARE may be embedded in a membrane that the vesicle will never encounter because its motor proteins or tethering proteins will not transport it there. Another method to achieve specificity is that the 4 a-helices can associate in several ways and due to their hydrophobic nature only a certain configuration will be thermodynamically stable enough to provide the energy for membrane fusion. But due to the high level of conservation in the SNARE domain of the SNARE families, it is hard to imagine that the associations themselves confer a large amount of specificity to the system. Indeed it is true that one SNARE can substitute another shown by SNAP-25 and SNAP-23 can substitute for each other in the exocytosis of chromaffin cells. One SNARE can also participate in several different fusion pathways for example VAMP8 is involved in late endosome fusion and also exocytosis in the pancreas. Another issue with the opinion that SNAREs confer specificity is that one type can often be found all over a membrane when a vesicle will only fuse with a particular section of the membrane. For example SNAREs Sso1p in yeast can be found all over the plasma membrane and yet vesicles only fuse with certain areas in polarised growth.

In conclusion I would say that tethering proteins and Ran confer the highest level of specificity during vesicle membrane fusion. Trafficking can provide some specificity in the direction and location that the vesicle is transported in. But it cannot protect against fusion with a membrane on the way to the final destination or indicate where exactly the vesicle should fuse. SNARE proteins offer a greater level of specificity as only some v and t SNAREs will associate in the correct, stable trans-SNARE formation. However SNAREs act at 25nm separation of vesicle and target membrane and tethering proteins act at 50nm and so I would say that tethering proteins are the most important stage. They ensure the vesicle leaves the cytoskeleton at the right place and ensure the correct vesicle makes contact with the correct SNARE protein.