Lecture 7 trafficking 2 Flashcards
What does SNARE stand for?
Soluble N-ethylmaleimide-sensitive fusion attachment receptor
How does the cell control which compartment a vesicle fuses with?
Addressing system with multiple components to increase specificity.
What do target membranes carry e.g., ER membrane or golgi?
Carry complementary markers.
What are the principle determinants of specificity?
SNAREs (R-SNAREs come from donor membrane and Q-SNAREs come from target compartments) and Rabs.
What are the characteristics of SNAREs?
At least 20 in an animal cell. They show little sequence homology only similar structural characteristics. Contain helical domains.
How do the helices of R and Q SNAREs interact?
Intertwine to form a stable trans-SNARE-complex. Difficult to separate them. This regulates transport and prevents unwanted vesicle fusion. The pairings between R and Q SNAREs is highly specific so vesicles dock in particular compartments.
What do Trans-SNARE-complexes form?
A 4-helix bundle. 1 helix is donated by the R-SNARE and 3 of the helices are donated by the Q-SNAREs.
What sequence pattern does SNAREs repeat?
A leucine zipper-like pattern. Q-SNAREs have an exclusively conserves glutamine residue. R-SNAREs have a conserved arginine. These are critical in the formation of the Trans-SNARE-complex. Mutations here has a severe effect on intracellular trafficking. Completely affects SNARE function.
What do leucine-zippers exhibit?
A repeating pattern of hydrophobic side chains every 7th residue.
What structure do SNARE 4-helix bundles adopt?
A layered structure that facilitates and stabilises strong interactions between SNARE helices. Example proteins include synaptobrevin (a R-SNARE 1 helix supplied), syntaxin (Q) supplies 1 helix, SNAP-25 (Q) supplies 2 helices. Similarities to leucine zipper.
What does the ionic interactions at layer 0 lead to?
Layer zero being completely shielded from the aqueous environment. Absolutely conserved arginine from R-SNARE forms three ionic interactions with each helix from Q-SNARE. Very stable. The helices that surround it completely block out any interactions with aqueous environment. If something could break this shield this interaction would fall apart. Some proteins that disassemble this may work by disrupting this shield to collapse the structure.
What would an R mutation such as an R-G substitution lead to?
Example in the yeast synaptobrevin homologue sec 22 is functionally equivalent to a sec22 deletion.
How are docking and fusion different?
Two separable events. Once docking has occurred fusion doesnt always occur immediately. Fusion required the approach of two membranes. SNAREs behave like a winch to pull the vesicle and acceptor membrane close together to fuse. Requires lots of energy.
Why does the pulling together of the vesicle and membrane require lots of energy?
Aqueous environment between vesicle and membrane. Most of the membranes involved have polar head groups. Have to drive the water away from the lipid head groups so they are close enough to fuse together.
How is this energy requirement achieved?
The trans-SNARE complex is very stable and upon binding releases lots of energy. Forms a stalk, hemifusion and then fusion.
How do SNAREs function in nerve cells?
As the vesicle arrives, synaptobrevin makes initial contact with the Q-SNAREs in the target membrane (the pre synaptic membrane). Synaptotagmin binds to calcium. Complexin binds to the trans-SNARE complex and prevents fusion from taking place. This is then primed ready to be released as a wave in response to a signal. Leads to calcium release which is sensed by synaptotagmin allows trans-SNARE complex to complete fusion and complexin is released.
What have viruses developed in a similar way to SNAREs?
A fusion apparatus similar to SNARE function. HIV has a surface protein called HIV fusion protein and it has a short hydrophobic end which docks to the Gp120 part of a receptor. This causes further docking events so HIV can insert into cell surface. Once this has occurred this complex collapses driving fusion into the cell. The structure of the collapsed HIV fusion protein is similar to the trans-SNARE complex structure but with 6 helices.
How are SNAREs recycled?
They must be separated for reuse. The NSF (N-ethylmaleimide-sensitive fusion) protein. N-ethylmaleimide is an alkylating agent and targets sulfhydryl groups. NSF cycles between the cytosol in an inactive form monomeric and an active polymeric form in the membrane. Two rings with 6 members of the protein in membrane (bi ring structure). Forms a complex with accessory proteins.
What family is NSF from?
ATPase member of the AAA family of ATPases. Chaperon proteins are also part of this family.
Why is SNARE disassembly required?
To prevent indiscriminate membrane fusion events occurring.
How does the structure of NSF link to its function?
Each member has ATPase domain. NSF has N domains that can recognise the accessory proteins which are called alpha SNAP (not same as the SNAP25). this acts as the link between NSF and the trans-SNARE complex.
What are the steps to disassembly?
alphaSNAP recognises the trans-SNARE through structural curvature of the membrane rather than by sequence. Recruits NSF via N domains. NSF binds ATP either sequential hydrolysis one by one or all at same time not sure. When the NSF binds to the SNAP complex the N domains move inwards and structure becomes compact. When ATP is hydrolysed that becomes reversed. N domains bound to SNAP move outwards. NSF is also moving away from membrane pulling trans-SNARE apart. So have both lateral movement and pulling force. The combination of this is enough to pull them apart to be recruited and reused.
