what are they used for?
What are SNARES for?
SNARES are proteins that mediate all of the trafficking steps of the secretory pathway. The proteins were first characterised in regulated exocytosis in neurons. Over 100 different SNARE proteins have been identified so far many of which have divergent amino acid sequences but overall they have a conserved mechanistic action.
SNARE classification
One way to classify SNAREs is by grouping them into T and V SNAREs based on the SNARE hypothesis. The SNARE hypothesis states that vesicles have V-SNAREs and targets have T-SNAREs hence the way the are named. The hypothesis suggests that when complementary T and V SNAREs are matched they facilitate membrane fusion.
The crystal structure of SNARE proteins has been elucidated for neurons of vertebrates:
- V- SNARE – made of 1 protein called synaptobrevin 2 (also know as VAMP which stands for vesicle associated membrane protein).
- T-SNARE – made of two proteins called SNAP-25 so named because it is a 25 kDa protein and Syntaxin 1.
www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=SNAREs&rid=cell.figgrp.3240 Follow this link for a diagram showing the role of V and T SNAREs in guiding vesicular traffic.
There are different isoforms of these three proteins that are used in other cells not just in neurons. Homologs have also been identified in yeast.
This system of classification is an oversimplification because a SNARE can be a T or a V SNARE in different environments. Also homotypic fusion events can occur so that two of the same type of SNARE can bond to one another to mediate fusion. Therefore the SNAREs are generally named by the membrane component they reside in.
The more accurate method of classifying SNAREs is to whether they contain glutamine(Q) or arginine(R). So a Q SNARE contains glutamine that contribute to the “0” layer in the central core complex and an R SNARE contains arginine. By this classification system synaptobrevin is an R SNARE and synatxin and SNAP 25 are Q SNAREs.
SNARE structure
Most vertebrate SNAREs have the following components:
- Simple domain structure
- SNARE motifs- conserved between all SNAREs and arranged in heptad repeats. These repeats are alternating hydrophobic and polar residues that are the basis for coiled coils and leucine zippers. A SNARE can have more than one snare motif for example SNAP 25 has two.
- C terminal transmembrane spanning domain – this is connected to the SNARE motif by a linker
- Hydrophobic posttranslational modifications – most SNAREs have these to mediate anchorage into the plasma membrane. For example SNAP-25 is palmitoylated(GLOSSARY).Its been discovered that palmitoylation confers protection from ubquitylation and subsequent degradation.
- N-terminus domain - Many SNAREs can also have a domain positioned at the N-terminus that can fold independently of other domains. These domains tend to be one of the most variable structural components of the SNAREs.
There are exceptions to the above description of a prototypical structure. The Brevins such as synaptobrevin lack the N-terminal transmembrane domain. And yeast SNAREs structure varies considerably compared to the vertebrate structure described above.
SNARE motifs
The interaction of the three SNARE proteins into complexes in mediated by SNARE motifs. When the SNAREs are not complexed the SNARE motifs are monomeric and unstructured however when the three SNARE motifs come together the motifs spontaenoulsy form a helical core complex which are extremely stable structures. These complexes consist of 4 helices, 2 αlpha-helices are contributed by SNAP-25 and 1 each from syntaxin and synaptobrevin. Each of the αlpha- helices is formed from a SNARE motif.
The centre of the helical core complex contains layers of interacting side chains many of which are hydrophobic except for a central layer “0” that has a highly conserved structure made up of glutamine(Q) and arginine(R) amino acid residues. The SNARE motifs can be classified based on whether they contain glutamine or arginine residues:
- Qa
- Qb
- Qc
- R-SNAREs
Functional SNARE complexes that drive membrane fusion are heterooligomeric meaning they have one of each of these of these. Therefore the complex can be referred to as a called a QabcR complex.
However because SNARE motifs are amphillic they can associate in combinations other than the functional QabcR complex. These complexes are less stable and it is believed they are non-functional because they may not have sufficient energy to drive membrane fusion. Neuronal SNAREs often have non-functional complexes.
N-Terminal domains
Q SNARES tend to have N-terminal domains that form anti-paralled three helix bundles that can vary in length. Whereas R-SNAREs have a profilin-like-folds sometimes referred to as longin domains ( profilin acts to prevent spontaneous actin polymerisation but it has also been suggested that it has a function in membrane trafficking and in lipid based signalling).
What is the function of N-terminal domains?
There are several suggections:
- SNARE closed conformation- some N-terminal domains act to keep a SNARE in a closed conformation so that it cannot form a SNARE complex. But not all act in this manner.
- Recruitment platforms – some N-terminal domains act as sites where other proteins can bind. For example SM proteins(sec1/munc18 related proteins) can bind in two ways. Arch shaped SM proteins can enclose and stabilise the SNARE in a closed conformation. Or the interaction can be confined to a surface interaction with the N-terminal domain on the SNARE only. SM proteins are essential for fusion.
- Fusion? - Some studies have suggested that some types of N-terminal domains are essential for fusion and some suggest fusion can occur independent of the presence of other types of N-terminal domain.
SNARE localisation
Eukaryotic cells contain many SNAREs at different locations, originally it was thought that there may be a specific set of SNAREs that could pair for each type of membrane fusion reaction. This is true to some extent some SNAREs only have one partner SNARE they can interact with to participate in a single type of fusion reaction e.g synaptobrevin only interacts with SNAP25 an syntaxin A or B. But some SNAREs have several sets of SNARE partners that they can interact with to mediate several types of fusion eg Endobrevin functions in late endosome fusion and in exocytosis from the pancreas.
Specifically localised SNAREs include:
- Plasma membrane – Syntaxin 1, syntaxin 2, sytaxin,4, SNAP23 and SNAP25.
- Synaptic/neurosecretory vesicles – synaptobrevin/ VAMP.
- Golgi – synaptobrevin 4, syntaxin 5.
Rab Proteins
Rab proteins are monomeric GTPases they are active in the GTP bound state and inactive in the GDP bound state. Conversion from GTP to GDP occurs by GAPS and conversion from GDP to GTP occurs by GEFs. There are over 30 known types that form a superfamily of proteins. Like SNAREs Rab proteins are localised to specific subcellular locations, c-termini swapping experiments have shown that this is how the rab proteins get localised to specific areas. Below are a few examples of where they are localised:
- Rab1 - ER and golgi complex.
- Rab 4 - early endosomes.
- Rab 9 - late endosomes and trans golgi network.
The purpose of Rab proteins is to pair SNAREs to add greater specifcity to the pairing. A rab-GTP on a vesicle binds to the correct rab effector on a target membrane, this allows the SNAREs to come in close proximity and pair with one another. Once fusion has occured the rab and rab effector dissociate when a GAP hyrdolyses the GTP bound to the Rab and it is recycled back to the membrane that the vesicle formed from. To ensure the rab doesnt dissocaite from the GDP GDI binds to the Rab-GDP.
www.steve.gb.com/images/science/rab_snare_targeting.png Follow this link to see a diagram on how SNAREs pair using Rabs.