Membrane Fusion

Question 1

1. Recognize the importance of sub-cellular protein targeting.

Answer 1

Vesicular transport is the basic principle of transporting substances within the cell from one compartment to another.
Incorrect transport can cause disease (CF due to failure in Cl- ion channel-protein transport).
The lipid bilayer has charged residues facing the outside (attached to the hydrophilic heads) and interacting with water molecules (because water has a dipole). But the presence of these charged molecules make fusion between membranes thermodynamically inefficient because there are repulsive ionic forces it needs to overcome. Also have to remove water before you fuse lipids. Sub-cellular protein targeting thus has machinery that increases efficiency of fusion that also ensures specificity.
Adhesion is the most difficult step, but then you get semi-fusion (the point of no return) and then fusion.
Motor proteins move along actin or microtubule tracks to help get transport vesicle where it needs to go, and helped by the SNAREs.

Question 2

2. Describe the basic principles of membrane and viral fusion, including:
   a) the function and structure of SNARE proteins

Answer 2

a) function and structure of SNARE proteins:
= 3 main classes of SNAREs =
-syntaxins:
syntaxin are in the target membrane
syntaxin have 1 conserved SNARE motif.
Syntaxin = transmembrane domain, also helical.

-VAMPSs (vesicle associated membrane protein):
VAMP is located in the transport vesicle
VAMP have 1 conserved SNARE motif.
VAMPS = transmembrane domain at one end, with a helical domain.

-SNAPs (synaptosome associated protein):
SNAP are in the target membrane.
SNAP has two conserved SNARE motifs.
These proteins interact to cause a fusion event (that is very stable).
SNAP25 = no transmembrane domain, 2 helical domains, and a fatty acid binding region (acts as a membrane-binding domain).

• Helices can bind with each other, form a ‘coil-coil’ motif and bring membranes close enough to overcome repulsive forces.
• VAMP helix will bind SNAP and syntaxin helices on target membrane (“zippering”) and press the layers together, squeeze out the water, and cause fusion.

Question 3

2. Describe the basic principles of membrane and viral fusion, including:
   b) regulation of SNARE-based fusion

Answer 3

b) regulation of SNARE-based fusion:
= NSF protein (an ATPase) sits on the SNARE complex and hydrolyzes ATP–> causes the complex to unwind and twist apart (with the help of alpha-SNAP).
-Now that syntaxin has been denatured in twisting process –> need n-sec-1 to help it refold properly into active conformation.
Syntaxin forms a pseudo-SNARE complex that makes it easier to interact with sec1.

Question 4

2. Describe the basic principles of membrane and viral fusion, including:
   c) the mechanism of viral fusion

Answer 4

c) mechanisms of viral fusion:
= Enveloped viruses (HIV, e-bola, influenza) have envelope outside of capsid that allows it to fuse membrane on host very similarly to SNARE fusion.
Viral fusion proteins have a transmembrane domain and a highly hydrophobic fusion domain (which is hidden inside the viral protein until it receives a signal.

Ex: in influenza, signal = drop in pH –> fusion domain gets exposed and is inserted into target cell membrane –> fusion event (using a 6-alpha helix complex). For HIV, the virus only fuses with CD4 cells because it binds the CD4 protein on cell membrane (via gp120) –> fusion. Target may not always be plasma membrane (such as influenza virus, which goes after endosome membrane after it gets into cell. When virus replicated, new envelope can be host-derived.

Question 5

2. Describe the basic principles of membrane and viral fusion, including:
   d) the regulation of viral fusion.

Answer 5

d) regulation of viral fusion:
= Activation step helps specify which kinds of cells a virus can infect. HIV entry inhibitors = a form of therapy –> drugs target the pocket forming domain (so that it can’t interact with pocket binding domain) and this somehow prevents a fusion event.