Lecture 13 - Vesicular trafficking 2

Question 1

Describe the role of adaptor proteins

Answer 1

• form an inner layer of proteins between the doner membrane where the vesicle will bud.
• recognise and select cargo ensuring specificity
• link the coat o the membrane
• adaptors recognise motifs (signals) in the cytoplasmic domains of membrane proteins

Question 2

How many related adaptor complexes are there?

Answer 2

5

Question 3

Describe the structure of adaptor proteins

Answer 3

All in one family due to similar structure - ‘inverted mickey mouse structure’ - made up of 2 large subunits, medium subunits and smaller subunits.

The large subunits have domains which are connected by flexible hinges. Each subunit have high homology to each other. The family of proteins were identified as having key roles in CARGO SELECTION.

Recognition site found on cargo, which are recognised by the adaptor proteins. This will then lead to them linking the cargo to the coat scaffold

Question 4

What adaptor proteins have important roles in neuronal cells?

Answer 4

AP-4 & 5

Question 5

Where is AP-2 found?

Answer 5

found in plasma membrane and can be detected via immunofluorescence

Question 6

Where is AP-1 found?

Answer 6

localised in the trans-Golgi network & endosomes

Question 7

Where is AP-3 found?

Answer 7

localized in trans-Golgi network

Question 7

What is the role of AP-3?

Answer 7

selects cargo to send to the axon

Question 7

Explain how different adaptors transport different cargo to different parts of the cell

Answer 7

• AP-3 - important for selecting cargo to send to the axon
• AP - 1/4 - important for selecting cargo into transport vesicles and sending them to dendrites
• AP-2 - at presynaptic terminal - important for endocytosis in clathrin-coated vesicles, therefore a key role in the retrieval of material which has been released during exocytosis at the presynapse
• AP-3 - important for generating synaptic vesicles
• AP-1 - taking material back to the cell body

Question 7

What is the role of AP-2?

Answer 7

Presynaptic terminal - endocytosis in clathrin-coated vesicles

Question 7

What is the role of AP-1/4?

Answer 7

important for selecting cargo into transport vesicles and sending them to dendrites

Question 8

What is the role of AP-3?

Answer 8

important for generating synaptic vesicles

Question 9

What is the role AP-1?

Answer 9

taking material back to the cell body

Question 10

What is a key point about adaptor proteins?

Answer 10

Although the adaptor proteins are structurally similar, they are very good at sorting cargo

Question 10

What subunits are important for recognizing the cargo?

Answer 10

medium and smaller subunits

Question 11

What are adaptor proteins important for?

Answer 11

ensuring the selectivity and fidelity of protein transport (ensuring the cargo goes to the right destination)

Question 12

Why is it useful that there are lots of ways proteins can be internalised?

Answer 12

This is useful due to the diversity of components that cells are exposed to in the environment

Question 13

What do Rab proteins regulate?

Answer 13

Specific trafficking steps

Question 14

How many Rab proteins are there?

Answer 14

Approximately 60-70 members in mammalian cells

Question 15

Where is Rab1 found?

Answer 15

Rab1 found in the golgi complex and plays a role in the fusion of incoming vesicles with a target membrane

Question 16

Describe features of Rab1

Answer 16

• member Ras superfamily (GTPases)
• distinct subcellular localization
• cycle between membrane and cytosol
• required for fusion

Question 17

What changes rabGDP to rabGTP?

Answer 17

rabGEFs

Question 18

What changes rabGTP to rabGDP?

Answer 18

rabGAPs

Question 19

What hydrolyses (off switch) rabGTP?

Answer 19

rabGAPs

Question 20

Describe the key steps of the Rab cycle

Answer 20

Rab-GTP + V-SNARE –> transport vesicle –> docking –> Rab effector + t-SNARE –> membrane fusion –> GTP hydrolysis –> GEF

Question 21

What are tethering proteins often?

Answer 21

Rab effectors - tethering –> docking –> fusion

Question 22

What does Rab5 regulate?

Answer 22

Regulates multiple events on the early endocytic pathway
- depending on where the cell is, it can utilize Rab cells differently

Question 25

What is a cranio-lenticulo-sutural dysplasia?

Answer 25

• autosomal recessive syndrome
• mutation in SEC23A
• connective tissue skeletal muscle disease - problems forming fontanels

Question 25

How does cranio-lenticulo-sutural dysplasia come about?

Answer 25

disease mutation causes aberrant ER to Golgi trafficking - visualized by immunofluorescence
- collagen is defective in these patients
- suggested that the underlying mechanism for this disease was a defect in the ER to Golgi trafficking

Question 25

What experiment allows us to understand what makes a COPII vesicle?

Answer 25

Reconstitution experiment

Question 25

What effect does a mutant SEC23A have in liposomes?

Answer 25

Binding of mutant SEC23A to liposomes is unaffected

Question 25

What does mutant SEC23A affect?

Answer 25

COPII coated vesicle formation is affected
- addition of purified coat proteins, even if the presence of low cytosol, you get a nice formation of COPII vesicles

Question 26

Explain what is affected in patients with cranio-lenticulo-sutural dysplasia (CLSD)

Answer 26

Patients with CLSD don’t have problems with digestion or insulin secretion. Only some tissues are affected as in mammalian cells, there are compensatory mechanisms, which allow the stomach and pancreas to overcome the defect.

Question 27

How does packaging of large cargo occur?

Answer 27

Large cargo fit in tubular structure, however have packaging proteins which help assembly in a particular way.

Question 28

What does mutations in Rab7 lead to?

Answer 28

• Charcot-Marie-Tooth neuropathy type 2B
• TrkA signalling is disrupted

Question 28

What is choroidermia?

Answer 28

Rabs and Rab-related proteins that causes blindness in men. Defect in CHM gene on X chromosome. Encodes for REP1. Gene therapy offers potential therapy. Adeno-associated virus delivers the defective gene

Question 28

What does mutations in Rab27a lead to?

Answer 28

• exocytosis of organelles
• Griscelli syndrome: haemophagocytic syndrome
• Albinism, delivery of melanin melanocytes

Question 29

What does mutations in Rab7 lead to?

Answer 29

• late endosomal trafficking
• Charcot-Marie-Tooth neuropathy type 2B
• TrkA signalling is disrupted