|Fig 1 life cycle of a synaptic vesicle|
- Vesicular trafficking in eukaryotic cells is essential for diverse cellular processes, including maintenance of distinct subcellular compartments, protein and hormone secretion, egg fertilization and neurotransmitter release
- the life cycle of a vesicle generally consists of three stages (
- endocytosis or formation of the vesicle from specific cellular membranes
- exocytosis or fusion of the vesicle with its target membran
- recycling of the components of the protein machinery after exocytosis.
- In synaptic vesicle exocytosis, three SNARE proteins are involved: the plasma-membrane-associated proteins syntaxin and SNAP-25 (synaptosomal-associated protein of 25kDa), and the vesicular protein synaptobrevin, also referred to as VAMP (vesicle-associated membrane protein).
- Other conserved proteins include the ATPase NSF and its adaptor, known as SNAP (soluble NSF-attachment protein), the Rab class of small G proteins and their effectors, the synaptotagmin family and the nSec1 (neuronal homolog of the yeast Sec1 protein, also referred to as Munc18) family.
- Initially, syntaxin is bound to nSec1 and synaptobrevin is probably bound to a factor such as synaptophysin or VAP33.
- yet to be identified molecular machinery probably brings the vesicle and plasma membrane into close proximity
- lets SNAREs on opposite membranes form cis complexes.
- Synaptobrevin then binds to syntaxin and SNAP-25
- At the priming stage, the system becomes competent to undergo fusion upon an increase in Ca2+ concentration in the micromolar range
- possibly involving a Ca2+-binding protein such as synaptotagmin
- At the recycling stage, α-SNAP and NSF bind to the SNARE complex,
- SNARE complex is then dissociated upon ATP hydrolysis.
- Before docking, vesicles have to be targeted to the correct location at the appropriate time.
- The SNARE core complex consists of a parallel four-helix bundle
- N-terminal domain of syntaxin consists of an antiparallel three-helix bundle
- the core of the four-helix bundle of the SNARE complex is composed of layers formed by interacting sidechains from each of the four α helices
- At centre of core complex, cnoserved ionic layer
- has arginine and 3 glutamines contributed by each of 4 alpha helices.
- Ionic layer is sealed off against solvent by adjacent hydrophobic layers.
- Mutations in these and other layers reduce complex stability
- cause defects in membrane trafficking
- SNAREs have several conformational states
- Closed conformation of uncomplexed syntaxin and unstructured or flexible comforms of synaptobrevin and SNAP-25
- Binary complex of syntaxin and SNAP-25
- Ternary complex of syntaxin, SNAP-25 and cytoplasmic domain of synaptobrevin
- Closed conformation of uncomplexed syntaxin contains a 4-helix bundle
- bundle made up of regulatory Nterminal HAHBHC domain and roughly half of the core complex domain Hcore
- A similar conformation of syntaxin has recently been observed in the crystal structure of syntaxin in the syntaxin–nSec1 complex
- may be the closed conformation of syntaxin that binds to nSec1.
- Syntaxin switches to an ‘open’ state upon binding to SNAP-25
- In this ‘open’ state, binding to the other SNAREs is mediated by the Hcore domain
Interactions of syntaxin with nSec1
- Partially structured, closed state of syntaxin interacts with nSec1.
- When complexed with nSec1, C terminal syntaxin residues unstructured or flexible in solution adopt a sequence of small a-helical frags connected by short loops
- In ternary SNARE complex these residues form a continuous a helix.
- Synaptotagmin is a membrane-associated protein that interacts with SNAREs, phospholipid membranes, Ca2+ channels and proteins involved in endocytosis
- In cytosolic portion, a flexible seven aa linkter joins 2 honomologous C2 domains, C2A and C2B.
- The C2A domain binds to anionic phospholipids and other accessory proteins, such as syntaxin
- in a Ca2+-dependent fashion.
- No conformational change is observed upon Ca2+ binding
- except for rotamer changes of the Ca2+-coordinating aspartic acid residues.
- The C2B domain promotes binding to other C2B domains as well as to accessory proteins, independently of Ca2+.
- Crystal structure of synaptotagmin II includes C2A and C2B domain
- Has differences in shape of Ca2+ binding pocket, electrostatic surface potential and stoichiometry of bound divalent cations for 2 domains
- Synaptotagmin covalently links 2 inndependent C2 domains, each with potentially different binding partners
- C2B is involved in synaptotagmin oligomerisation
- Synaptotagmin and SNARE complex interact indepent of Ca2+
- Ca2+ enhances interaction
- Ca2+ binding domains interact with plasma membrane