membrane fusion 1

What is membrane fusion

The merging of a transport vesicle membrane with a target membrane to allow cargo delivery

What are the two main challenges in membrane fusion

Specificity of fusion and overcoming the energetic barrier

What is the major energetic barrier to membrane fusion

The hydration barrier caused by water bound to charged polar lipid head groups

How close must membranes come to overcome the hydration barrier

Approximately 1 nanometer

Why does membrane fusion require proteins

Lipids alone cannot overcome the hydration and lipid tail energy barriers

What two biological systems illustrate membrane fusion

Transport vesicles in cells and enveloped viruses

Why are viral fusion proteins useful models

They show how protein conformational energy can drive membrane fusion

Which influenza protein mediates membrane fusion

Hemagglutinin

Which HIV protein directly mediates membrane fusion

gp41

What triggers conformational change in viral fusion proteins

Binding to host cell receptors

What is a fusion peptide

A hydrophobic peptide that inserts into the target membrane during fusion

Why is the fusion peptide compared to a transmembrane domain

It is hydrophobic and inserts into lipid bilayers

What structural change drives viral membrane fusion

Formation of a hairpin composed of alpha helices

How does the viral hairpin promote fusion

By pulling the viral and host membranes together

What secondary structure dominates viral fusion proteins

Alpha helices

What is a coiled-coil

A structure formed by multiple alpha helices packing via hydrophobic stripes

How many degrees does each residue advance in an alpha helix

Approximately 100 degrees

Which amino acid positions form hydrophobic stripes in coiled-coils

Positions 1 4 and 7

Why are coiled-coils energetically important

Their high stability releases energy that drives membrane fusion

What experimental system demonstrates fast cellular fusion

Mast cell exocytosis

How is membrane fusion detected electrophysiologically

By measuring increases in membrane capacitance

What does membrane capacitance reflect

The surface area of the membrane

What is the timescale of membrane fusion in cells

Approximately one millisecond

What property of fusion shows it can be reversible

Fusion pores can open and close repeatedly

Who reconstituted intra Golgi transport biochemically

Rothman and colleagues

Why is in vitro reconstitution important

It allows biochemical manipulation and identification of fusion factors

What reagent was used to identify fusion proteins

N ethylmaleimide NEM

How does NEM inhibit fusion

By modifying essential cysteine residues in proteins

What did cytosol addition after NEM treatment show

A cytosolic factor is required for fusion

What protein was identified as NEM sensitive

NSF

What type of protein is NSF

A AAA ATPase and unfoldase

How fast does NSF hydrolyze ATP

Approximately one ATP per second

Why can NSF not directly drive fast fusion

Its ATPase activity is too slow

What protein allows NSF to bind membranes

SNAP

What does SNAP stand for

Soluble NSF Attachment Protein

Is NSF required in all trafficking pathways

Yes it is ubiquitous

Why are synaptic vesicles a useful model system

Their components are well characterized and fusion is fast

What triggers synaptic vesicle fusion

Calcium influx following an action potential

What three proteins were identified as SNAP receptors

Syntaxin SNAP 25 and synaptobrevin

What are these proteins collectively called

SNAREs

Which SNARE is located on vesicles

Synaptobrevin also called VAMP

Which SNAREs are on the target membrane

Syntaxin and SNAP 25

What type of membrane anchoring do SNAREs have

C terminal anchors or lipid modification

What structure do SNAREs form together

A stable trimeric coiled coil complex

What provided functional proof SNAREs are essential

Clostridial neurotoxins such as botulinum toxin

How do botulinum toxins inhibit neurotransmission

By cleaving SNARE proteins

Why must SNAREs be compartment specific

To ensure selective membrane fusion

What was the original SNARE hypothesis

ATP hydrolysis by NSF drives membrane fusion

Why was the original SNARE hypothesis rejected

Fusion is too fast and reversible for NSF driven ATP hydrolysis

What did SNARE crystal structures reveal

They resemble viral fusion hairpins

What feature of SNAREs provides energy for fusion

The stability of the coiled coil complex

How was SNARE mediated fusion proven experimentally

By liposome fusion assays

How is fusion detected in liposome assays

By fluorescence dequenching due to lipid dilution

What fluorescent dye is commonly used

BODIPY

What happens to fluorescence upon membrane fusion

It increases due to dequenching

What control shows fusion is SNARE specific

v SNARE and t SNARE liposomes fuse but v v do not

Why does botulinum toxin fail after SNARE complex formation

The stable complex is inaccessible to cleavage

What is the revised role of NSF and SNAP

To disassemble and recycle SNARE complexes after fusion

Why must SNARE complexes be disassembled

So SNAREs can be reused for future fusion events

What class of proteins does NSF belong to

AAA ATPase unfoldases

What other cellular machines share this function

Vps4 and the proteasome regulatory cap

What ultimately drives membrane fusion

Energy released from SNARE complex zippering