Lecture 13 - Vesicular trafficking 2 Flashcards
Describe the role of adaptor proteins
- 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
How many related adaptor complexes are there?
5
Describe the structure of adaptor proteins
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
What adaptor proteins have important roles in neuronal cells?
AP-4 & 5
Where is AP-2 found?
found in plasma membrane and can be detected via immunofluorescence
Where is AP-1 found?
localised in the trans-Golgi network & endosomes
Where is AP-3 found?
localized in trans-Golgi network
What is the role of AP-3?
selects cargo to send to the axon
Explain how different adaptors transport different cargo to different parts of the cell
- 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
What is the role of AP-2?
Presynaptic terminal - endocytosis in clathrin-coated vesicles
What is the role of AP-1/4?
important for selecting cargo into transport vesicles and sending them to dendrites
What is the role of AP-3?
important for generating synaptic vesicles
What is the role AP-1?
taking material back to the cell body
What is a key point about adaptor proteins?
Although the adaptor proteins are structurally similar, they are very good at sorting cargo
What subunits are important for recognizing the cargo?
medium and smaller subunits
What are adaptor proteins important for?
ensuring the selectivity and fidelity of protein transport (ensuring the cargo goes to the right destination)
Why is it useful that there are lots of ways proteins can be internalised?
This is useful due to the diversity of components that cells are exposed to in the environment
What do Rab proteins regulate?
Specific trafficking steps
How many Rab proteins are there?
Approximately 60-70 members in mammalian cells
Where is Rab1 found?
Rab1 found in the golgi complex and plays a role in the fusion of incoming vesicles with a target membrane
Describe features of Rab1
- member Ras superfamily (GTPases)
- distinct subcellular localization
- cycle between membrane and cytosol
- required for fusion
What changes rabGDP to rabGTP?
rabGEFs
What changes rabGTP to rabGDP?
rabGAPs
What hydrolyses (off switch) rabGTP?
rabGAPs
Describe the key steps of the Rab cycle
Rab-GTP + V-SNARE –> transport vesicle –> docking –> Rab effector + t-SNARE –> membrane fusion –> GTP hydrolysis –> GEF
What are tethering proteins often?
Rab effectors - tethering –> docking –> fusion
What does Rab5 regulate?
Regulates multiple events on the early endocytic pathway
- depending on where the cell is, it can utilize Rab cells differently
What is a cranio-lenticulo-sutural dysplasia?
- autosomal recessive syndrome
- mutation in SEC23A
- connective tissue skeletal muscle disease - problems forming fontanels
How does cranio-lenticulo-sutural dysplasia come about?
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
What experiment allows us to understand what makes a COPII vesicle?
Reconstitution experiment
What effect does a mutant SEC23A have in liposomes?
Binding of mutant SEC23A to liposomes is unaffected
What does mutant SEC23A affect?
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
Explain what is affected in patients with cranio-lenticulo-sutural dysplasia (CLSD)
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.
How does packaging of large cargo occur?
Large cargo fit in tubular structure, however have packaging proteins which help assembly in a particular way.
What does mutations in Rab7 lead to?
- Charcot-Marie-Tooth neuropathy type 2B
- TrkA signalling is disrupted
What is choroidermia?
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
What does mutations in Rab27a lead to?
- exocytosis of organelles
- Griscelli syndrome: haemophagocytic syndrome
- Albinism, delivery of melanin melanocytes
What does mutations in Rab7 lead to?
- late endosomal trafficking
- Charcot-Marie-Tooth neuropathy type 2B
- TrkA signalling is disrupted
