Membrane Trafficking

Question 1

What are SNAREs important for and which approaches were taken to identify them?

Answer 1

Synaptic vesicle fusion, Secretion of serum proteins, mucus secretion & intracellular transport of proteins.

Biochemical reconstitution, Yeast genetics & cloning

Question 2

What were Rothman’s hypothesis and were they correct?

Answer 2

SNARE for every transport step within a cell
SNAREs should provide specificity to vesicle transport
SNAREs should be sufficient to drive bilayer fusion
Proposed that NSF & ATP hydrolysis catalysed membrane fusion - this is incorrect.

Question 3

Tell me about the process of SNARE zippering

Answer 3

1. Trans-SNARE complex: Tight SNARE complexes form, ‘vesicle docking’
2. Hemifusion of outer lipid bilayer
3. Inner lipid layer fuses creating a fusion pore opening
4. Cis-SNARE complex: V-SNARES detach from vesicle & fusion pore enlarges
5. From here, membrane fusion is energetically favourable, energy provided by SNARE proteins

Question 4

What is the Q/R residue?

Answer 4

SNAREs can be divided into either Q or R SNAREs depending on whether they have an Arginine (R) residue at the 0 layer or a Glutamate (Q) residue at the 0 layer. 3Q interact with 1R, this is thought to be critical to SNARE disassembly, mutation of the Q/R residue inhibits SNARE activity.
The 3Q:1R ratio is conserved across evolution. Humans have 27Q SNAREs and 9 R SNAREs

Question 5

Tell me about SNARE specificity

Answer 5

Fusion only occurs in SNARE complexes that fit a 3Q:1R ratio.
SNARE’s do show some promiscuity but predominantly interact with SNAREs from the appropriate membrane.
Lot’s of additional machinery contributes towards SNARE specificity such as rabs, coats, and tethers.

Question 6

What are common features of SNAREs?

Answer 6

VAMP (V-SNARE) on the vesicle
SNAP & Syntaxin on the membrane. SNAPs have two coiled-coil domains
Generally small, 14-40kDa & all contain at least one SNARE motif (coiled-coil)
Generally C-terminal anchored

Question 7

Tell me about NSF

Answer 7

NSF recycles SNAREs after fusion. Uses ATP hydrolysis to unwind cis-SNARE complexes. When NSF is bound, considered as a transient 20s NSF/SNARE/SNAP complex.

Question 8

Tell me about SNARE mutations in mice

Answer 8

VAMP2 & SNAP25: Mice die at birth due a loss of synaptic transmission
Syntaxin 1A: No gross abnormalities. Subtle defects in synaptic transmission
Syntaxin 1b: Die after birth due to reduced synaptic transmission

Question 9

Tell me about VAMP mutation in humans

Answer 9

VAMP2 mutation leads to a neurodevelopmental disorder with hypotonia and autistic features with or without hyperkinetic movements.
Mutation S75P in VAMP2 V-SNARE domain slows the rate of liposome fusion. It is a dominant negative mutation.

Question 10

Tell me about SNAP mutations in humans

Answer 10

SNAP25b leads to a neurodevelopmental disorder with seizures, intellectual disability, severe speech delay and cerebellar ataxia.

SNAP29 leads to cerebral dysgenesis, neuropathy, icthyosis & palmoplantar keratoderma syndrone (CEDNIK)

Question 11

Tell me about FHL4

Answer 11

Familial Hemophagocytic Lymphohistiocytosis 4 can be caused by many genetic mutations. One such mutation is a mutation in the Q-SNARE of Syntaxin11 (STX11) which results in reduced STX11 levels.
FHL4 involves the over-proliferation of T cells, NK cells, B cells and macrophages. Patients can die of infection as a result of defective B-cell killing.
Mutation in Munc18-2 causes FHL5 & reduced STX11 levels

Question 12

Tell me about clostridial neurotoxins

Answer 12

Clostridium tetani causes tetanus through tetanus neurotoxins which cleaves VAMPs at inhibitory neurons leading to rigid muscles until a painful death, causes ~50,000 deaths a year.
Clostridium botulinum causes botulism, it inhibits neuromuscular junctions (NMJs), infant botulism is the most common form - ‘floppy baby syndrome’. 100-200 cases per year with 1-2 deaths.

Question 13

Describe the structure and function of botulinum toxin

Answer 13

Has a targeting domain which binds to neurons via endosome action
Has a translocation domain which pokes pores in the endosome to release the proteasome domain
Has a proteasome domain which cleaves specific SNAREs

Question 14

Give some examples of commercial uses for botulinum toxin

Answer 14

Cosmetic uses - Botox
Medical uses, in parkinsons it can prevent oversalivation, muscle spams and improve bladder control
BoNT/A has many uses such as in Botox, Pain relief, reduces chronic pain
BoNT/B can treat cervical dystonia.

Question 15

Tell me about the nuclear pore complex and diffusion through it

Answer 15

Made up of ~30 nucleoporins, a central plug and other subunits. Free diffusion can occur with proteins upto 5,000Da.
For proteins upto 17,000Da, it takes 30 minutes to reach equilibrium and 2 hours for proteins upto 44,000Da. Upto 60,000Da proteins require active transport.

Question 16

Tell me about active transport through the NPC

Answer 16

The NPC can open upto 26nm in diameter. Actively transported proteins require an NLS: Nuclear Localisation Sequence. NLS’s are usually peptide sequences, SV40 virus has the NLS of PPKKKRKV.

There is experimental evidence suggesting that active transport into the nucleus requires ATP hydrolysis. ATP hydrolysis is inhibited at 4°C, when SV40 was stained and the conditions were 4°C, it wasn’t localised to the nucleus, however, it was bound to NPCs.

Question 17

Tell me about ER translocation

Answer 17

When mRNAs are translocated, the newly synthesized proteins are translocated into the ER lumen via a translocator protein, this requires that the ribosomes are closely associated to ER, e.g., RER.

Question 18

What is signal hypothesis?

Answer 18

The translocator opens when it binds a start-transfer signal sequence which is usually a peptide sequence. The translocator binds the signal sequence and the newly synthesized polypeptide is looped into the ER lumen. Signal peptidases in the ER lumen cleave the signal sequence closing the translocator.

Membrane bound proteins contain stop-transfer sequences that can close the translocator mid-translation. The stop-transfer sequences are usually very hydrophobic, key in membrane anchoring.

Proteins with multiple transmembrane domains contain multiple start/stop transfer sequences.

Question 19

What is the difference between type 1 and type 2 membrane protein topology?

Answer 19

Type 1: N-terminus faces the cytosol, C-terminus faces the extracellular space. Immediately after translation, the N-terminus faces the cytosol and C-terminus faces the ER lumen.

Type 2: N-terminus faces the extracellular space, C-terminus faces the cytosol. Immediately after translation, the N-terminus faces the ER lumen and C-terminus faces the cytosol.

Question 20

Tell me about chaperones in the ER

Answer 20

Chaperones in the ER are responsible for protein folding.
BiP associates with newly synthesized proteins to ensure proper folding. Many quality control measures exist:
a) misfolding results in reverse translocation and degradation in the cytoplasm.
b) Glycosylation occurs in the ER & Golgi, key in quality control
c) Misfolded proteins stimulate the UPR - unfolded protein response, occurs in plants with different machinery

Defects in protein misfolding lead to disease: CFTRΔ508 leads to cystic fibrosis.

Question 21

Tell me about protein translocation in mitochondria

Answer 21

N-terminus sequence is recognised by TOM complex: peptides translocated across MOM (Mitochondrial Outer Membrane). Chaperones assist protein folding in the intermembrane space. SAM inserts the proteins into the MOM where as TIM23 translocates membranes across the MIM (Mitochondrial Inner Membrane) after which the signal is cleaved by a signal peptidase.

The signal is an amphipathic α-helix which binds to a receptor that recognises structure rather than specific residues with polar residues binding in polar grooves.

Question 22

Tell me about protein translocation in bacteria

Answer 22

Sec import polypeptides into the periplasm, periplasmic chaperones assist folding and BAM inserts them into the outer membrane.

Question 23

Tell me about protein translocation in chloroplasts

Answer 23

Uses TOC and TIC complexes.

Question 24

Describe the process of nuclear import

Answer 24

1. Importins bind to cargo via NLS
2. Importin/Cargo diffuses into the nucleus
3. Ran/GTP binds to Importin/Cargo stimulating cargo release
4. Ran/GTP/Importin hydrolysed to Ran/GDP/Importin in cytoplasm by Ran/GAP
5. Importin is released

Question 25

Describe the process of COPII vesicle formation

Answer 25

1. Cargo receptor recruits Sec24
2. Sec24 recruits Sec23
3. Sec23 recruits Sar1 which is activated by ER membrane-bound GEF Sec12
4. Sar1-GTP recruits Sec13/31, the outer protein coat

Question 26

Tell me about coat disassembly

Answer 26

Sec23 is the GAP for Sar1, it’s GAP activity is enhanced by Sec13/31 which stimulates the disassembly of the protein coat. The protein coat is required to stabilise the budding event but inhibits vesicle membrane fusion so has to be removed after budding.

Question 27

Tell me about the discovery of COPII vesicle proteins

Answer 27

This was done via a reductionist reconstitution experiment where cell components such as the cytosol, ATP and GTP were added back to the cell.
ER and Vesicles were isolated via centrifuge action and run on a gel to detect the presence of ribophorin, an ER protein which should be isolated to the ER and p58 a commonly exported protein which should be present in the ER.
They found that cytosol, ATP and GTP were required for proper vesicle formation and that from the cytosol, Sar1, Sec23/24/13/31 were required alongside ER membrane bound Sec12.

Question 28

What are some common vesicles and their properties?

Answer 28

1. COPII vesicles contain a COPII protein coat, controlled by Sar1 GTPase and carry newly synthesized proteins
2. COPI vesicles contain a COPI protein coat, controlled by Arf GTPase and carry retrieved proteins and newly synthesized proteins
3. Clathrin (Trans-golgi-network) vesicles contain a clathrin coat, controlled by Arf GTPase and carry lysosomal proteins and regulatory secreted proteins
4. Clathrin (plasma-membrane) vesicles contain a clathrin coat, controlled by an unknown GTPase and carry endocytosed material

Question 29

What is CLSD?

Answer 29

Cranio-leticulo-sutural disease occurs due to a F -> K mutation in Sec23A. It results in fontanels not closing, skeletal defects, hypermotility and muscular skeletal issues. The extent of ER distension depends on the whether it is a heterozygous or homozygous mutation. The coat isn’t recruited to COPII vesicles.

Reconstitution experiments were used to understand this. Liposome binding assays showed that coat proteins can still bind to GTP-Sar1; Sec23A mutation doesn’t impact coat binding to Sar and COPII vesicles weren’t being contaminated with ER proteins

Why Sec23 mutations result in reduced vesicle formation is largely unknown. Zebrafish used as a model demonstrate that mutant Sec23 does retain some function and can be compensated for by Sec23B.

The reduced transport of collagen is a key cause of the muscular skeletal issues.

Question 30

What is Charcot-Marie-Tooth 2B?

Answer 30

Rab7a missense mutation makes it constitutively active leading to reduced autophagic flux, premature neurotrophin receptor degradation, inhibition of neurite growth

Question 31

How does Legionella pneumophila use rab1?

Answer 31

It recruits rab1 to the cell surface to create an ER-like compartment it can replicate in.

Question 32

Tell me about the Rab family of GTPases

Answer 32

The Rab family has over 60 members and is part of the Ras superfamily. Usually Rabs are found in the cytosol and recruited to membranes: they are key in fusion & other trafficking functions. Most members are not tissue-specific but ubiquitously expressed. Each Rab is associated with a specific membrane.

Rabs interact with tethering proteins, pulling Vesicles in close enough for SNARE action. Rab cascades allow the movement of cargo between organelles. One Rab can act as a GEF or another Rab.

Question 33

What is the secretory/exocytic pathway?

Answer 33

ER -> Golgi -> Plasma membrane OR endosomes OR lysosome (vacuole in plants/yeast).
Allows the constitutive transport/secretion of proteins

Question 34

What were the approaches used by Novick & Schekmans’ Sec screen in 1980?

Answer 34

They mutagenised cells and selected for cells which didn’t secrete invertase & acid phosphatase at permissive and restrictive temperatures (simply assay using the enzymes). Electron microscopy was used to observe changes in cell ultrastructure such as the accumulation of vesicles or aberrant membranes. 23 genes were identified, classed depending on the impact they had.

Question 35

What is a class A mutant gene:

Answer 35

Secreted proteins accumulate in the cytosol due to defective transport into the ER

Question 36

What is a class B mutant gene:

Answer 36

Secreted proteins accumulate in the RER due to defective budding from the RER

Question 37

What is a class C mutant gene:

Answer 37

Secreted proteins accumulate in the ER-golgi transport vesicles due to defective fusion of transport vesicles with the golgi

Question 38

What is a class D mutant gene:

Answer 38

Secreted proteins accumulate in the golgi due to defective transport from golgi to secretory vesicles

Question 39

What is a class E mutant gene:

Answer 39

Secreted proteins accumulate in secretory vesicles due to defective transport from secretory vesicles to the cell surface

Question 40

What are some of the problems with the Sec screens?

Answer 40

Only considered transport to the plasma membrane, not transport to endosomes or the vacuole. Any redundancy wouldn’t have been detected. Not all mutated genes became temperature dependent.